Methods of treating cancer using pd-1 axis binding antagonists and tigit inhibitors

ABSTRACT

The present invention describes combination treatment comprising a PD-1 axis binding antagonist and an agent that decreases or inhibits TIGIT expression and/or activity and methods for use thereof, including methods of treating conditions where enhanced immunogenicity is desired such as increasing tumor immunogenicity for the treatment of cancer or chronic infection.

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. ProvisionalApplication No. 61/846,941, filed Jul. 16, 2013, U.S. ProvisionalApplication No. 61/865,582, filed Aug. 13, 2013, U.S. ProvisionalApplication No. 61/950,754, filed Mar. 10, 2014, U.S. ProvisionalApplication No. 61/985,884, filed Apr. 29, 2014, and U.S. ProvisionalApplication No. 61/992,109, filed May 12, 2014, each of which is herebyincorporated by reference in its entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name:146392025900SEQLISTING.TXT, date recorded: Jul. 16, 2014, size: 25 KB).

BACKGROUND OF THE INVENTION

The provision of two distinct signals to T-cells is a widely acceptedmodel for lymphocyte activation of resting T lymphocytes byantigen-presenting cells (APCs). Lafferty et al, Aust. J. Exp. Biol.Med. ScL 53: 27-42 (1975). This model further provides for thediscrimination of self from non-self and immune tolerance. Bretscher etal, Science 169: 1042-1049 (1970); Bretscher, P. A., P.N.A.S. USA 96:185-190 (1999); Jenkins et al, J. Exp. Med. 165: 302-319 (1987). Theprimary signal, or antigen specific signal, is transduced through theT-cell receptor (TCR) following recognition of foreign antigen peptidepresented in the context of the major histocompatibility-complex (MHC).The second or co-stimulatory signal is delivered to T-cells byco-stimulatory molecules expressed on antigen-presenting cells (APCs),and induce T-cells to promote clonal expansion, cytokine secretion andeffector function. Lenschow et al., Ann. Rev. Immunol. 14:233 (1996). Inthe absence of co-stimulation, T-cells can become refractory to antigenstimulation, which results in a tolerogenic response to either foreignor endogenous antigens.

In the two-signal model, T-cells receive both positive co-stimulatoryand negative co-inhibitory signals. The regulation of such positive andnegative signals is critical to maximize the host's protective immuneresponses, while maintaining immune tolerance and preventingautoimmunity. Negative signals seem necessary for induction of T-celltolerance, while positive signals promote T-cell activation.

Both co-stimulatory and co-inhibitory signals are provided toantigen-exposed T cells, and the interplay between co-stimulatory andco-inhibitory signals is essential to controlling the magnitude of animmune response. Further, the signals provided to the T cells change asan infection or immune provocation is cleared, worsens, or persists, andthese changes powerfully affect the responding T cells and re-shape theimmune response.

The mechanism of co-stimulation is of therapeutic interest because themanipulation of co-stimulatory signals has shown to provide a means toeither enhance or terminate cell-based immune response. Recently, it hasbeen discovered that T cell dysfunction or anergy occurs concurrentlywith an induced and sustained expression of the inhibitory receptor,programmed death 1 polypeptide (PD-1). As a result, therapeutictargeting of PD-1 and other molecules which signal through interactionswith PD-1, such as programmed death ligand 1 (PD-L1) and programmeddeath ligand 2 (PD-L2) are an area of intense interest.

PD-L1 is overexpressed in many cancers and is often associated with poorprognosis (Okazaki T et al., Intern. Immun. 2007 19(7):813) (Thompson RH et al., Cancer Res 2006, 66(7):3381). Interestingly, the majority oftumor infiltrating T lymphocytes predominantly express PD-1, in contrastto T lymphocytes in normal tissues and peripheral blood T lymphocytesindicating that up-regulation of PD-1 on tumor-reactive T cells cancontribute to impaired antitumor immune responses (Blood 2009114(8):1537). This may be due to exploitation of PD-L1 signalingmediated by PD-L1 expressing tumor cells interacting with PD-1expressing T cells to result in attenuation of T cell activation andevasion of immune surveillance (Sharpe et al., Nat Rev 2002) (Keir M Eet al., 2008 Annu. Rev. Immunol. 26:677). Therefore, inhibition of thePD-L1/PD-1 interaction may enhance CD8+ T cell-mediated killing oftumors.

The inhibition of PD-1 axis signaling through its direct ligands (e.g.,PD-L1, PD-L2) has been proposed as a means to enhance T cell immunityfor the treatment of cancer (e.g., tumor immunity). Moreover, similarenhancements to T cell immunity have been observed by inhibiting thebinding of PD-L1 to the binding partner B7-1. Furthermore, combininginhibition of PD-1 signaling with other signaling pathways that arederegulated in tumor cells may further enhance treatment efficacy. Thereremains a need for such an optimal therapy for treating, stabilizing,preventing, and/or delaying development of various cancers.

All references, publications, and patent applications disclosed hereinare hereby incorporated by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

The present invention describes a combination treatment comprising aPD-1 axis binding antagonist and an agent that decreases or inhibitsTIGIT expression and/or activity.

Provided herein are methods for treating or delaying progression ofcancer in an individual comprising administering to the individual aneffective amount of a PD-1 axis binding antagonist and an agent thatdecreases or inhibits TIGIT expression and/or activity.

Provided herein are also methods for reducing or inhibiting cancerrelapse or cancer progression in an individual comprising administeringto the individual an effective amount of a PD-1 axis binding antagonistand an agent that decreases or inhibits TIGIT expression and/oractivity.

Provided herein are also methods for treating or delaying progression ofan immune related disease in an individual comprising administering tothe individual an effective amount of a PD-1 axis binding antagonist andan agent that decreases or inhibits TIGIT expression and/or activity.

Provided herein are also methods for reducing or inhibiting progressionof an immune related disease in an individual comprising administeringto the individual an effective amount of a PD-1 axis binding antagonistand an agent that decreases or inhibits TIGIT expression and/oractivity.

In some embodiments, the immune related disease is associated with a Tcell dysfunctional disorder. In some embodiments, the T celldysfunctional disorder is characterized by decreased responsiveness toantigenic stimulation. In some embodiments, the T cell dysfunctionaldisorder is characterized by T cell anergy or decreased ability tosecrete cytokines, proliferate or execute cytolytic activity. In someembodiments, the T cell dysfunctional disorder is characterized by Tcell exhaustion. In some embodiments, the T cells are CD4+ and CD8+ Tcells. In some embodiments, the immune related disease is selected fromthe group consisting of unresolved acute infection, chronic infectionand tumor immunity.

Provided herein are also methods of increasing, enhancing or stimulatingan immune response or function in an individual by administering to theindividual an effective amount of a PD-1 axis binding antagonist and anagent that decreases or inhibits TIGIT expression and/or activity.

Provided herein are also methods of treating or delaying progression ofcancer in an individual comprising administering to the individual aneffective amount of a PD-1 axis binding antagonist and an agent thatmodulates the CD226 expression and/or activity.

Provided herein are also methods for reducing or inhibiting cancerrelapse or cancer progression in an individual comprising administeringto the individual an effective amount of a PD-1 axis binding antagonistand an agent that modulates the CD226 expression and/or activity.

Provided herein are also methods for treating or delaying progression ofan immune related disease in an individual comprising administering tothe individual an effective amount of a PD-1 axis binding antagonist andan agent that modulates the CD226 expression and/or activity.

Provided herein are also methods for reducing or inhibiting progressionof an immune related disease in an individual comprising administeringto the individual an effective amount of a PD-1 axis binding antagonistand agent that modulates the CD226 expression and/or activity.

In some embodiments, the immune related disease is associated with a Tcell dysfunctional disorder. In some embodiments, the T celldysfunctional disorder is characterized by decreased responsiveness toantigenic stimulation. In some embodiments, the T cell dysfunctionaldisorder is characterized by T cell anergy, or decreased ability tosecrete cytokines, proliferate or execute cytolytic activity. In someembodiments, the T cell dysfunctional disorder is characterized by Tcell exhaustion. In some embodiments, the T cells are CD4+ and CD8+ Tcells. In some embodiments, the immune related disease is selected fromthe group consisting of unresolved acute infection, chronic infectionand tumor immunity.

Provided herein are also methods of increasing, enhancing or stimulatingan immune response or function in an individual by administering to theindividual an effective amount of a PD-1 axis binding antagonist and anagent that modulates the CD226 expression and/or activity.

In some embodiments, the agent that modulates the CD226 expressionand/or activity is capable of increasing and/or stimulating CD226expression and/or activity.

In some embodiments, the agent that modulates the CD226 expressionand/or activity is selected from an agent that inhibits and/or blocksthe interaction of CD226 with TIGIT, an antagonist of TIGIT expressionand/or activity, an antagonist of PVR expression and/or activity, anagent that inhibits and/or blocks the interaction of TIGIT with PVR, anagent that inhibits and/or blocks the intracellular signaling mediatedby TIGIT binding to PVR.

In some embodiments, the agent that inhibits and/or blocks theinteraction of CD226 with TIGIT is a small molecule inhibitor, aninhibitory antibody or antigen-binding fragment thereof, an aptamer, aninhibitory nucleic acid, and an inhibitory polypeptide. In someembodiments, the agent that inhibits and/or blocks the interaction ofCD226 with TIGIT is an anti-TIGIT antibody or antigen-binding fragmentthereof.

In some embodiments, the antagonist of TIGIT expression and/or activityis a small molecule inhibitor, an inhibitory antibody or antigen-bindingfragment thereof, an aptamer, an inhibitory nucleic acid, and aninhibitory polypeptide. In some embodiments, the antagonist of TIGITexpression and/or activity is an anti-TIGIT antibody or antigen-bindingfragment thereof.

In some embodiments, the antagonist of PVR expression and/or activity isa small molecule inhibitor, an inhibitory antibody or antigen-bindingfragment thereof, an aptamer, an inhibitory nucleic acid, and aninhibitory polypeptide.

In some embodiments, the agent that inhibits and/or blocks theinteraction of TIGIT with PVR is a small molecule inhibitor, aninhibitory antibody or antigen-binding fragment thereof, an aptamer, aninhibitory nucleic acid, and an inhibitory polypeptide.

In some embodiments, the agent that inhibits and/or blocks theintracellular signaling mediated by TIGIT binding to PVR is a smallmolecule inhibitor, an inhibitory antibody or antigen-binding fragmentthereof, an aptamer, an inhibitory nucleic acid, and an inhibitorypolypeptide.

The present invention also describes a combination treatment comprisingan agent that decreases or inhibits TIGIT expression and/or activity andan agent that decreases or inhibits one or more additional immuneco-inhibitory receptors.

Provided herein are methods of increasing, enhancing or stimulating animmune response or function in an individual by administering to theindividual an effective amount of an agent that decreases or inhibitsTIGIT expression and/or activity and an agent that decreases or inhibitsone or more additional immune co-inhibitory receptors.

In some embodiments, the one or more additional immune co-inhibitoryreceptor is selected from the group consisting of PD-1, CTLA-4, LAG3,TIM3, BTLA and VISTA. In some embodiments, the one or more additionalimmune co-inhibitory receptor is selected from the group of PD-1,CTLA-4, LAG3 and TIM3.

The present invention also describes a combination treatment comprisingan agent that decreases or inhibits TIGIT expression and/or activity andan agent that increases or activates one or more additional immuneco-stimulatory receptor.

Provided herein are methods of increasing, enhancing or stimulating animmune response or function in an individual by administering to theindividual an effective amount of an agent that decreases or inhibitsTIGIT expression and/or activity and an agent that increases oractivates one or more additional immune co-stimulatory receptor.

In some embodiments, the one or more additional immune co-stimulatoryreceptor is selected from the group consisting of CD226, OX-40, CD28,CD27, CD137, HVEM, and GITR. In some embodiments, the one or moreadditional immune co-stimulatory receptor is selected from the group ofCD226, OX-40, CD27, CD137, HVEM and GITR. In some embodiments, the oneor more additional immune co-stimulatory receptor is selected from thegroup consisting of OX-40 and CD27.

In some embodiments, any of the above methods further compriseadministering at least one chemotherapeutic agent.

In some embodiments, the individual in any of the above methods hascancer. In some embodiments, the individual in any of the above methodsis a human.

In some embodiments, the CD4 and/or CD8 T cells in the individual haveincreased or enhanced priming, activation, proliferation, cytokinerelease and/or cytolytic activity relative to prior to theadministration of the combination.

In some embodiments, the number of CD4 and/or CD8 T cells is elevatedrelative to prior to administration of the combination. In someembodiments, the number of activated CD4 and/or CD8 T cells is elevatedrelative to prior to administration of the combination. In someembodiments, the activated CD4 and/or CD8 T cells is characterized byγ-IFN⁺ producing CD4 and/or CD8 T cells and/or enhanced cytolyticactivity relative to prior to the administration of the combination. Insome embodiments, the CD4 and/or CD8 T cells exhibit increased releaseof cytokines selected from the group consisting of IFN-γ, TNF-α, andinterleukins.

In some embodiments, the CD4 and/or CD8 T cell is an effector memory Tcell. In some embodiments, the CD4 and/or CD8 effector memory T cell ischaracterized by γ-IFN⁺ producing CD4 and/or CD8 T cells and/or enhancedcytolytic activity. In some embodiments, the CD4 and/or CD8 effectormemory T cell is characterized by having the expression of CD44^(high)CD62L^(low).

In some embodiments, the cancer in any of the above methods has elevatedlevels of T cell infiltration.

In some embodiments, the agent that decreases or inhibits TIGITexpression and/or activity is selected from the group consisting of anantagonist of TIGIT expression and/or activity, an antagonist of PVRexpression and/or activity, and an agent that inhibits the interactionand/or the intracellular signaling mediated by TIGIT binding to PVR.

In some embodiments, the antagonist of TIGIT expression and/or activityis selected from the group consisting of a small molecule inhibitor, aninhibitory antibody or antigen-binding fragment thereof, an aptamer, aninhibitory nucleic acid, and an inhibitory polypeptide.

In some embodiments, the antagonist of PVR expression and/or activity isselected from the group consisting of a small molecule inhibitor, aninhibitory antibody or antigen-binding fragment thereof, an aptamer, aninhibitory nucleic acid, and an inhibitory polypeptide.

In some embodiments, the agent that inhibits the intracellular signalingmediated by TIGIT binding to PVR is selected from the group consistingof a small molecule inhibitor, an inhibitory antibody or antigen-bindingfragment thereof, an aptamer, an inhibitory nucleic acid, and aninhibitory polypeptide.

In some embodiments, the antagonist of TIGIT expression and/or activityis an anti-TIGIT antibody or antigen-binding fragment thereof.

In some embodiments, the anti-TIGIT antibody or antigen-binding fragmentthereof comprises at least one HVR comprising an amino acid sequenceselected from the amino acid sequences KSSQSLYYSGVKENLLA (SEQ ID NO:1),ASIRFT (SEQ ID NO:2), QQGINNPLT (SEQ ID NO:3), GFTFSSFTMH (SEQ ID NO:4),FIRSGSGIVFYADAVRG (SEQ ID NO:5), and RPLGHNTFDS (SEQ ID NO:6) orRSSQSLVNSYGNTFLS (SEQ ID NO:7), GISNRFS (SEQ ID NO:8), LQGTHQPPT (SEQ IDNO:9), GYSFTGHLMN (SEQ ID NO:10), LIIPYNGGTSYNQKFKG (SEQ ID NO:11), andGLRGFYAMDY (SEQ ID NO:12).

In some embodiments, the anti-TIGIT antibody or antigen-binding fragmentthereof comprises a light chain comprising the amino acid sequence setforth in DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTK LEIKR (SEQID NO:13) orDVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPPTFGPGTKLEVK (SEQ ID NO:14).

In some embodiments, the anti-TIGIT antibody or antigen-binding fragmentthereof comprises a heavy chain comprising the amino acid sequence setforth in EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVT VSS (SEQ IDNO:15) or EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGLIIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGLRGFYAMDYWGQGTS VT VSS (SEQ IDNO:16).

In some embodiments, the anti-TIGIT antibody or antigen-binding fragmentthereof comprises a light chain comprising the amino acid sequence setforth in DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTK LEIKR (SEQID NO:13) orDVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPPTFGPGTKLEVK (SEQ ID NO:14) andthe antibody heavy chain comprises the amino acid sequence set forth inEVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVT VSS (SEQ IDNO:15) or EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGLIIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGLRGFYAMDYWGQGTSVT VSS (SEQ IDNO:16).

In some embodiments, the anti-TIGIT antibody or antigen-binding fragmentthereof, wherein the antibody is selected from a humanized antibody, achimeric antibody, a bispecific antibody, a heteroconjugate antibody,and an immunotoxin.

In some embodiments, the anti-TIGIT antibody or antigen-binding fragmentthereof comprises at least one HVR is at least 90% identical to an HVRset forth in any of KSSQSLYYSGVKENLLA (SEQ ID NO:1), ASIRFT (SEQ IDNO:2), QQGINNPLT (SEQ ID NO:3), GFTFSSFTMH (SEQ ID NO:4),FIRSGSGIVFYADAVRG (SEQ ID NO:5), and RPLGHNTFDS (SEQ ID NO:6) orRSSQSLVNSYGNTFLS (SEQ ID NO:7), GISNRFS (SEQ ID NO:8), LQGTHQPPT (SEQ IDNO:9), GYSFTGHLMN (SEQ ID NO:10), LIIPYNGGTSYNQKFKG (SEQ ID NO:11), andGLRGFYAMDY (SEQ ID NO:12).

In some embodiments, the anti-TIGIT antibody or fragment thereofcomprises the light chain and/or heavy chain comprising amino acidsequences at least 90% identical to the amino acid sequences set forthin DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTK LEIKR (SEQID NO:13) orDVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPPTFGPGTKLEVK (SEQ ID NO:14), orEVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVT VSS (SEQ IDNO:15) or EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGLIIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGLRGFYAMDYWGQGTSVT VSS (SEQ IDNO:16), respectively.

In some embodiments, the PD-1 axis binding antagonist is selected fromthe group consisting of a PD-1 binding antagonist, a PD-L1 bindingantagonist and a PD-L2 binding antagonist.

In some embodiments, the PD-1 axis binding antagonist is a PD-1 bindingantagonist. In some embodiments, the PD-1 binding antagonist inhibitsthe binding of PD-1 to its ligand binding partners. In some embodiments,the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1. Insome embodiments, the PD-1 binding antagonist inhibits the binding ofPD-1 to PD-L2. In some embodiments, the PD-1 binding antagonist inhibitsthe binding of PD-1 to both PD-L1 and PD-L2. In some embodiments, thePD-1 binding antagonist is an antibody. In some embodiments, the PD-1binding antagonist is MDX-1106 (nivolumab). In some embodiments, thePD-1 binding antagonist is Merck 3475 (lambrolizumab). In someembodiments, the PD-1 binding antagonist is CT-011 (pidilizumab). Insome embodiments, the PD-1 binding antagonist is AMP-224.

In some embodiments, the PD-1 axis binding antagonist is a PD-L1 bindingantagonist. In some embodiments, the PD-L1 binding antagonist inhibitsthe binding of PD-L1 to PD-1. In some embodiments, the PD-L1 bindingantagonist inhibits the binding of PD-L1 to B7-1. In some embodiments,the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1and B7-1. In some embodiments, the PD-L1 binding antagonist is anantibody.

In some embodiments, the PD-L1 binding antagonist is selected from thegroup consisting of: YW243.55.S70, MPDL3280A, MDX-1105 and MEDI 4736.

In some embodiments, the anti-PD-L1 antibody comprises a heavy chaincomprising HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO:17), HVR-H2 sequenceof AWISPYGGSTYYADSVKG (SEQ ID NO:18), and HVR-H3 sequence of RHWPGGFDY(SEQ ID NO:19); and a light chain comprising HVR-L1 sequence ofRASQDVSTAVA (SEQ ID NO:20), HVR-L2 sequence of SASFLYS (SEQ ID NO:21),and HVR-L3 sequence of QQYLYHPAT (SEQ ID NO:22).

In some embodiments, the anti-PD-L1 antibody comprises a heavy chainvariable region comprising the amino acid sequence ofEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVT VSA (SEQ IDNO:23) and a light chain variable region comprising the amino acidsequence of DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO:24).

In some embodiments, the PD-1 axis binding antagonist is a PD-L2 bindingantagonist. In some embodiments, the PD-L2 binding antagonist is anantibody. In some embodiments, the PD-L2 binding antagonist is animmunoadhesin.

In some embodiments, the cancer being treated is selected from the groupconsisting of non-small cell lung cancer, small cell lung cancer, renalcell cancer, colorectal cancer, ovarian cancer, breast cancer,pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer,mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma,prostate cancer, glioblastoma, cervical cancer, thymic carcinoma,leukemia, lymphomas, myelomas, mycoses fungoids, merkel cell cancer, andother hematologic malignancies.

In some embodiments, the agent that decreases or inhibits TIGITexpression and/or activity is administered continuously. In someembodiments, the agent that decreases or inhibits TIGIT expressionand/or activity is administered intermittently. In some embodiments, theagent that decreases or inhibits TIGIT expression and/or activity isadministered before the PD-1 axis binding antagonist. In someembodiments, the agent that decreases or inhibits TIGIT expressionand/or activity is administered simultaneous with the PD-1 axis bindingantagonist. In some embodiments, the agent that decreases or inhibitsTIGIT expression and/or activity is administered after the PD-1 axisbinding antagonist.

Also provided herein are kits comprising a PD-1 axis binding antagonistand a package insert comprising instructions for using the PD-1 axisbinding antagonist in combination with an agent that decreases orinhibits TIGIT expression and/or activity to treat or delay progressionof cancer in an individual.

Also provided herein are kits comprising a PD-1 axis binding antagonistand an agent that decreases or inhibits TIGIT expression and/oractivity, and a package insert comprising instructions for using thePD-1 axis binding antagonist and the agent that decreases or inhibitsTIGIT expression and/or activity to treat or delay progression of cancerin an individual.

Also provided herein are kits comprising an agent that decreases orinhibits TIGIT expression and/or activity and a package insertcomprising instructions for using the agent that decreases or inhibitsTIGIT expression and/or activity in combination with a PD-1 axis bindingantagonist to treat or delay progression of cancer in an individual.

Also provided herein are kits comprising a PD-1 axis binding antagonistand a package insert comprising instructions for using the PD-1 axisbinding antagonist in combination with an agent that decreases orinhibits TIGIT expression and/or activity to enhance immune function ofan individual having cancer.

Also provided herein are kits comprising a PD-1 axis binding antagonistand an agent that decreases or inhibits TIGIT expression and/oractivity, and a package insert comprising instructions for using thePD-1 axis binding antagonist and the agent that decreases or inhibitsTIGIT expression and/or activity to enhance immune function of anindividual having cancer.

Also provided herein are kits comprising an agent that decreases orinhibits TIGIT expression and/or activity and a package insertcomprising instructions for using the agent that decreases or inhibitsTIGIT expression and/or activity in combination with a PD-1 axis bindingantagonist to enhance immune function of an individual having cancer.

Also provided herein are kits comprising a PD-1 axis binding antagonistand a package insert comprising instructions for using the PD-1 axisbinding antagonist in combination with an agent that modulates the CD226expression and/or activity to treat or delay progression of cancer in anindividual.

Also provided herein are kits comprising a PD-1 axis binding antagonistand an agent that modulates the CD226 expression and/or activity, and apackage insert comprising instructions for using the PD-1 axis bindingantagonist and the agent that modulates the CD226 expression and/oractivity to treat or delay progression of cancer in an individual.

Also provided herein are kits comprising an agent that modulates theCD226 expression and/or activity and a package insert comprisinginstructions for using the agent modulates the CD226 expression and/oractivity in combination with a PD-1 axis binding antagonist to treat ordelay progression of cancer in an individual.

Also provided herein are kits comprising a PD-1 axis binding antagonistand a package insert comprising instructions for using the PD-1 axisbinding antagonist in combination with an agent that modulates the CD226expression and/or activity to enhance immune function of an individualhaving cancer.

Also provided herein are kits comprising a PD-1 axis binding antagonistand an agent that modulates the CD226 expression and/or activity, and apackage insert comprising instructions for using the PD-1 axis bindingantagonist and the agent that modulates the CD226 expression and/oractivity to enhance immune function of an individual having cancer.

Also provided herein are kits comprising an agent modulates the CD226expression and/or activity and a package insert comprising instructionsfor using the agent that modulates the CD226 expression and/or activityin combination with a PD-1 axis binding antagonist to enhance immunefunction of an individual having cancer.

In some embodiments, the kits comprising the PD-1 axis bindingantagonist is an anti-PD-L1 antibody. In some embodiments, the kitscomprising the PD-1 axis binding antagonist is an anti-PD-1 antibody. Insome embodiments, the kits comprising the agent that decreases orinhibits TIGIT expression and/or activity is selected from the groupconsisting of an antagonist of TIGIT expression and/or activity, anantagonist of PVR expression and/or activity, and an agent that inhibitsthe interaction and/or the intracellular signaling mediated by TIGITbinding to PVR. In some embodiments, the kits comprising the antagonistof TIGIT expression and/or activity is an anti-TIGIT antibody orantigen-binding fragment thereof.

In some embodiments, the kits comprises an agent that modulates theCD226 expression and/or activity which is capable of increasing and/orstimulating CD226 expression and/or activity. In some embodiments, thekits comprising the agent that modulates the CD226 expression and/oractivity is selected from an agent that inhibits and/or blocks theinteraction of CD226 with TIGIT, an antagonist of TIGIT expressionand/or activity, an antagonist of PVR expression and/or activity, anagent that inhibits and/or blocks the interaction of TIGIT with PVR, anagent that inhibits and/or blocks the intracellular signaling mediatedby TIGIT binding to PVR. In some embodiments, the kits comprising theagent that inhibits and/or blocks the interaction of CD226 with TIGITand/or the antagonist of TIGIT expression and/or activity is ananti-TIGIT antibody or antigen-binding fragment thereof.

In certain aspects, the present disclosure provides a method fortreating or delaying progression of cancer in an individual comprisingadministering to the individual an effective amount of a PD-1 axisbinding antagonist and an agent that decreases or inhibits TIGITexpression and/or activity. In other aspects, the present disclosureprovides use of an effective amount of a PD-1 axis binding antagonist inthe manufacture of a medicament for treating or delaying progression ofcancer in an individual, wherein the PD-1 axis binding agent is used incombination with an agent that decreases or inhibits TIGIT expressionand/or activity. In other aspects, the present disclosure provides useof an effective amount of an agent that decreases or inhibits TIGITexpression and/or activity in the manufacture of a medicament fortreating or delaying progression of cancer in an individual, wherein thean agent that decreases or inhibits TIGIT expression and/or activity isused in combination with a PD-1 axis binding antagonist. In otheraspects, the present disclosure provides a pharmaceutical compositioncomprising a PD-1 axis binding antagonist for use in treating ordelaying progression of cancer in combination with an agent thatdecreases or inhibits TIGIT expression and/or activity. In otheraspects, the present disclosure provides a pharmaceutical compositioncomprising an agent that decreases or inhibits TIGIT expression and/oractivity for use in treating or delaying progression of cancer incombination with a PD-1 axis binding antagonist.

In other aspects, the present disclosure provides a method for reducingor inhibiting cancer relapse or cancer progression in an individualcomprising administering to the individual an effective amount of a PD-1axis binding antagonist and an agent that decreases or inhibits TIGITexpression and/or activity. In other aspects, the present disclosureprovides use of an effective amount of a PD-1 axis binding antagonist inthe manufacture of a medicament for reducing or inhibiting cancerrelapse or cancer progression in an individual, wherein the PD-1 axisbinding agent is used in combination with an agent that decreases orinhibits TIGIT expression and/or activity. In other aspects, the presentdisclosure provides use of an effective amount of an agent thatdecreases or inhibits TIGIT expression and/or activity in themanufacture of a medicament for reducing or inhibiting cancer relapse orcancer progression in an individual, wherein the agent that decreases orinhibits TIGIT expression and/or activity is used in combination with aPD-1 axis binding antagonist. In other aspects, the present disclosureprovides a pharmaceutical composition comprising a PD-1 axis bindingantagonist for use in reducing or inhibiting cancer relapse or cancerprogression in combination with an agent that decreases or inhibitsTIGIT expression and/or activity. In other aspects, the presentdisclosure provides a pharmaceutical composition comprising an agentthat decreases or inhibits TIGIT expression and/or activity for use inreducing or inhibiting cancer relapse or cancer progression incombination with a PD-1 axis binding antagonist.

In other aspects, the present disclosure provides a method for treatingor delaying progression of an immune related disease in an individualcomprising administering to the individual an effective amount of a PD-1axis binding antagonist and an agent that decreases or inhibits TIGITexpression and/or activity. In other aspects, the present disclosureprovides use of an effective amount of a PD-1 axis binding antagonist inthe manufacture of a medicament for treating or delaying progression ofan immune related disease in an individual, wherein the PD-1 axisbinding agent is used in combination with an agent that decreases orinhibits TIGIT expression and/or activity. In other aspects, the presentdisclosure provides use of an effective amount of an agent thatdecreases or inhibits TIGIT expression and/or activity in themanufacture of a medicament for treating or delaying progression of animmune related disease in an individual, wherein the agent thatdecreases or inhibits TIGIT expression and/or activity is used incombination with a PD-1 axis binding antagonist. In other aspects, thepresent disclosure provides a pharmaceutical composition comprising aPD-1 axis binding antagonist for use in treating or delaying progressionof an immune related disease in combination with an agent that decreasesor inhibits TIGIT expression and/or activity. In other aspects, thepresent disclosure provides a pharmaceutical composition comprising anagent that decreases or inhibits TIGIT expression and/or activity foruse in treating or delaying progression of an immune related disease incombination with a PD-1 axis binding antagonist.

In other aspects, the present disclosure provides a combinationcomprising an effective amount of a PD-1 axis binding antagonist and anagent that decreases or inhibits TIGIT expression and/or activity.

In other aspects, the present disclosure provides a method for reducingor inhibiting progression of an immune related disease in an individualcomprising administering to the individual an effective amount of a PD-1axis binding antagonist and an agent that decreases or inhibits TIGITexpression and/or activity. In other aspects, the present disclosureprovides use of an effective amount of a PD-1 axis binding antagonist inthe manufacture of a medicament for reducing or inhibiting progressionof an immune related disease in an individual, wherein the PD-1 axisbinding agent is used in combination with an agent that decreases orinhibits TIGIT expression and/or activity. In other aspects, the presentdisclosure provides use of an effective amount of an agent thatdecreases or inhibits TIGIT expression and/or activity in themanufacture of a medicament for reducing or inhibiting progression of animmune related disease in an individual, wherein the agent thatdecreases or inhibits TIGIT expression and/or activity is used incombination with a PD-1 axis binding antagonist. In other aspects, thepresent disclosure provides a pharmaceutical composition comprising aPD-1 axis binding antagonist for use in reducing or inhibitingprogression of an immune related disease in combination with an agentthat decreases or inhibits TIGIT expression and/or activity. In otheraspects, the present disclosure provides a pharmaceutical compositioncomprising an agent that decreases or inhibits TIGIT expression and/oractivity for use in reducing or inhibiting progression of an immunerelated disease in combination with a PD-1 axis binding antagonist.

In certain embodiments that may be combined with any of the precedingembodiments, the immune related disease is associated with a T celldysfunctional disorder. In certain embodiments that may be combined withany of the preceding embodiments, the immune related disease is a viralinfection. In certain embodiments that may be combined with any of thepreceding embodiments, the viral infection is a chronic viral infection.In certain embodiments that may be combined with any of the precedingembodiments, the T cell dysfunctional disorder is characterized bydecreased responsiveness to antigenic stimulation. In certainembodiments that may be combined with any of the preceding embodiments,the T cell dysfunctional disorder is characterized by T cell anergy ordecreased ability to secrete cytokines, proliferate or execute cytolyticactivity. In certain embodiments that may be combined with any of thepreceding embodiments, the T cell dysfunctional disorder ischaracterized by T cell exhaustion. In certain embodiments that may becombined with any of the preceding embodiments, the T cells are CD4+ andCD8+ T cells. In certain embodiments that may be combined with any ofthe preceding embodiments, the immune related disease is selected fromthe group consisting of unresolved acute infection, chronic infection,and tumor immunity.

In other aspects, the present disclosure provides a method ofincreasing, enhancing or stimulating an immune response or function inan individual comprising administering to the individual an effectiveamount of a PD-1 axis binding antagonist and an agent that decreases orinhibits TIGIT expression and/or activity. In other aspects, the presentdisclosure provides use of an effective amount of a PD-1 axis bindingantagonist in the manufacture of a medicament for enhancing orstimulating an immune response or function in an individual, wherein thePD-1 axis binding agent is used in combination with an agent thatdecreases or inhibits TIGIT expression and/or activity. In otheraspects, the present disclosure provides use of an effective amount ofan agent that decreases or inhibits TIGIT expression and/or activity inthe manufacture of a medicament for enhancing or stimulating an immuneresponse or function in an individual, wherein the agent that decreasesor inhibits TIGIT expression and/or activity is used in combination witha PD-1 axis binding antagonist. In other aspects, the present disclosureprovides a pharmaceutical composition comprising a PD-1 axis bindingantagonist for use in enhancing or stimulating an immune response orfunction in combination with an agent that decreases or inhibits TIGITexpression and/or activity. In other aspects, the present disclosureprovides a pharmaceutical composition comprising an agent that decreasesor inhibits TIGIT expression and/or activity for use in enhancing orstimulating an immune response or function in combination with a PD-1axis binding antagonist. In other aspects, the present disclosureprovides a combination comprising an effective amount of a PD-1 axisbinding antagonist and an agent that decreases or inhibits TIGITexpression and/or activity.

In other aspects, the present disclosure provides a method of treatingor delaying progression of cancer in an individual comprisingadministering to the individual an effective amount of a PD-1 axisbinding antagonist and an agent that modulates CD226 expression and/oractivity. In other aspects, the present disclosure provides use of aneffective amount of a PD-1 axis binding antagonist in the manufacture ofa medicament for treating or delaying progression of cancer in anindividual, wherein the PD-1 axis binding agent is used in combinationwith an agent that modulates CD226 expression and/or activity. In otheraspects, the present disclosure provides use of an effective amount ofan agent that modulates CD226 expression and/or activity in themanufacture of a medicament for treating or delaying progression ofcancer in an individual, wherein the agent that modulates CD226expression and/or activity is used in combination with a PD-1 axisbinding antagonist. In other aspects, the present disclosure provides apharmaceutical composition comprising a PD-1 axis binding antagonist foruse in treating or delaying progression of cancer in combination with anagent that modulates CD226 expression and/or activity. In other aspects,the present disclosure provides a pharmaceutical composition comprisingan agent that modulates CD226 expression and/or activity for use intreating or delaying progression of cancer in combination with a PD-1axis binding antagonist.

In other aspects, the present disclosure provides a method for reducingor inhibiting cancer relapse or cancer progression in an individualcomprising administering to the individual an effective amount of a PD-1axis binding antagonist and an agent that modulates CD226 expressionand/or activity. In other aspects, the present disclosure provides useof an effective amount of a PD-1 axis binding antagonist in themanufacture of a medicament for reducing or inhibiting cancer relapse orcancer progression in an individual, wherein the PD-1 axis binding agentis used in combination with an agent that modulates CD226 expressionand/or activity. In other aspects, the present disclosure provides useof an effective amount of an agent that modulates CD226 expressionand/or activity in the manufacture of a medicament for reducing orinhibiting cancer relapse or cancer progression in an individual,wherein the agent that modulates CD226 expression and/or activity isused in combination with a PD-1 axis binding antagonist. In otheraspects, the present disclosure provides a pharmaceutical compositioncomprising a PD-1 axis binding antagonist for use in reducing orinhibiting cancer relapse or cancer progression in combination with anagent that modulates CD226 expression and/or activity. In other aspects,the present disclosure provides a pharmaceutical composition comprisingan agent that modulates CD226 expression and/or activity for use inreducing or inhibiting cancer relapse or cancer progression incombination with a PD-1 axis binding antagonist.

In other aspects, the present disclosure provides a method for treatingor delaying progression of an immune related disease in an individualcomprising administering to the individual an effective amount of a PD-1axis binding antagonist and an agent that modulates CD226 expressionand/or activity. In other aspects, the present disclosure provides useof an effective amount of a PD-1 axis binding antagonist in themanufacture of a medicament for treating or delaying progression of animmune related disease in an individual, wherein the PD-1 axis bindingagent is used in combination with an agent that modulates CD226expression and/or activity. In other aspects, the present disclosureprovides use of an effective amount of an agent that modulates CD226expression and/or activity in the manufacture of a medicament fortreating or delaying progression of an immune related disease in anindividual, wherein the agent that modulates CD226 expression and/oractivity is used in combination with a PD-1 axis binding antagonist. Inother aspects, the present disclosure provides a pharmaceuticalcomposition comprising a PD-1 axis binding antagonist for use intreating or delaying progression of an immune related disease incombination with an agent that modulates CD226 expression and/oractivity. In other aspects, the present disclosure provides apharmaceutical composition comprising an agent that modulates CD226expression and/or activity for use in treating or delaying progressionof an immune related disease in combination with a PD-1 axis bindingantagonist.

In other aspects, the present disclosure provides a combinationcomprising an effective amount of a PD-1 axis binding antagonist and anagent that modulates CD226 expression and/or activity.

In other aspects, the present disclosure provides a method for reducingor inhibiting progression of an immune related disease in an individualcomprising administering to the individual an effective amount of a PD-1axis binding antagonist and an agent that modulates CD226 expressionand/or activity. In other aspects, the present disclosure provides useof an effective amount of a PD-1 axis binding antagonist in themanufacture of a medicament for reducing or inhibiting progression of animmune related disease in an individual, wherein the PD-1 axis bindingagent is used in combination with an agent that modulates CD226expression and/or activity. In other aspects, the present disclosureprovides use of an effective amount of an agent that modulates CD226expression and/or activity in the manufacture of a medicament forreducing or inhibiting progression of an immune related disease in anindividual, wherein the agent that modulates CD226 expression and/oractivity is used in combination with a PD-1 axis binding antagonist. Inother aspects, the present disclosure provides a pharmaceuticalcomposition comprising a PD-1 axis binding antagonist for use inreducing or inhibiting progression of an immune related disease incombination with an agent that modulates CD226 expression and/oractivity. In other aspects, the present disclosure provides apharmaceutical composition comprising an agent that modulates CD226expression and/or activity for use in reducing or inhibiting progressionof an immune related disease in combination with a PD-1 axis bindingantagonist.

In certain embodiments that may be combined with any of the precedingembodiments, the immune related disease is associated with a T celldysfunctional disorder. In certain embodiments that may be combined withany of the preceding embodiments, the immune related disease is a viralinfection. In certain embodiments that may be combined with any of thepreceding embodiments, the viral infection is a chronic viral infection.In certain embodiments that may be combined with any of the precedingembodiments, the T cell dysfunctional disorder is characterized bydecreased responsiveness to antigenic stimulation. In certainembodiments that may be combined with any of the preceding embodiments,the T cell dysfunctional disorder is characterized by T cell anergy, ordecreased ability to secrete cytokines, proliferate or execute cytolyticactivity. In certain embodiments that may be combined with any of thepreceding embodiments, the T cell dysfunctional disorder ischaracterized by T cell exhaustion. In certain embodiments that may becombined with any of the preceding embodiments, the T cells are CD4+ andCD8+ T cells. In certain embodiments that may be combined with any ofthe preceding embodiments, the immune related disease is selected fromthe group consisting of unresolved acute infection, chronic infectionand tumor immunity.

In other aspects, the present disclosure provides a method ofincreasing, enhancing, or stimulating an immune response or function inan individual comprising administering to the individual an effectiveamount of a PD-1 axis binding antagonist and an agent that modulatesCD226 expression and/or activity. In other aspects, the presentdisclosure provides use of an effective amount of a PD-1 axis bindingantagonist in the manufacture of a medicament for enhancing orstimulating an immune response or function in an individual, wherein thePD-1 axis binding agent is used in combination with an agent thatmodulates CD226 expression and/or activity. In other aspects, thepresent disclosure provides use of an effective amount of an agent thatmodulates CD226 expression and/or activity in the manufacture of amedicament for enhancing or stimulating an immune response or functionin an individual, wherein the an agent that modulates CD226 expressionand/or activity is used in combination with a PD-1 axis bindingantagonist. In other aspects, the present disclosure provides apharmaceutical composition comprising a PD-1 axis binding antagonist foruse in enhancing or stimulating an immune response or function incombination with an agent that modulates CD226 expression and/oractivity. In other aspects, the present disclosure provides apharmaceutical composition comprising an agent that modulates CD226expression and/or activity for use in enhancing or stimulating an immuneresponse or function in combination with a PD-1 axis binding antagonist.In other aspects, the present disclosure provides a combinationcomprising an effective amount of a PD-1 axis binding antagonist and anagent that modulates CD226 expression and/or activity.

In certain embodiments that may be combined with any of the precedingembodiments, the agent that modulates CD226 expression and/or activityis an agent that increases and/or stimulates CD226 expression and/oractivity. In certain embodiments that may be combined with any of thepreceding embodiments, the agent that modulates CD226 expression and/oractivity is an agent that increases and/or stimulates the interaction ofCD226 with PVR. In certain embodiments that may be combined with any ofthe preceding embodiments, the agent that modulates CD226 expressionand/or activity is an agent that increases and/or stimulates theintracellular signaling mediated by CD226 binding to PVR. In certainembodiments that may be combined with any of the preceding embodiments,the agent that modulates CD226 expression and/or activity is selectedfrom the group consisting of an agent that inhibits and/or blocks theinteraction of CD226 with TIGIT, an antagonist of TIGIT expressionand/or activity, an antagonist of PVR expression and/or activity, anagent that inhibits and/or blocks the interaction of TIGIT with PVR, anagent that inhibits and/or blocks the interaction of TIGIT with PVRL2,an agent that inhibits and/or blocks the interaction of TIGIT withPVRL3, an agent that inhibits and/or blocks the intracellular signalingmediated by TIGIT binding to PVR, an agent that inhibits and/or blocksthe intracellular signaling mediated by TIGIT binding to PVRL2, an agentthat inhibits and/or blocks the intracellular signaling mediated byTIGIT binding to PVRL3, and combinations thereof. In certain embodimentsthat may be combined with any of the preceding embodiments, the agentthat modulates CD226 expression and/or activity is an agent thatinhibits and/or blocks the interaction of CD226 with TIGIT. In certainembodiments that may be combined with any of the preceding embodiments,the agent that inhibits and/or blocks the interaction of CD226 withTIGIT is a small molecule inhibitor, an inhibitory antibody orantigen-binding fragment thereof, an aptamer, an inhibitory nucleicacid, or an inhibitory polypeptide. In certain embodiments that may becombined with any of the preceding embodiments, the agent that inhibitsand/or blocks the interaction of CD226 with TIGIT is an anti-TIGITantibody or antigen-binding fragment thereof. In certain embodimentsthat may be combined with any of the preceding embodiments, the agentthat inhibits and/or blocks the interaction of CD226 with TIGIT is aninhibitory nucleic acid selected from the group consisting of anantisense polynucleotide, an interfering RNA, a catalytic RNA, and anRNA-DNA chimera. In certain embodiments that may be combined with any ofthe preceding embodiments, the antisense polynucleotide targets TIGIT.In certain embodiments that may be combined with any of the precedingembodiments, the interfering RNA targets TIGIT. In certain embodimentsthat may be combined with any of the preceding embodiments, thecatalytic RNA targets TIGIT. In certain embodiments that may be combinedwith any of the preceding embodiments, the RNA-DNA chimera targetsTIGIT. In certain embodiments that may be combined with any of thepreceding embodiments, the agent that modulates CD226 expression and/oractivity is an antagonist of TIGIT expression and/or activity. Incertain embodiments that may be combined with any of the precedingembodiments, the antagonist of TIGIT expression and/or activity is asmall molecule inhibitor, an inhibitory antibody or antigen-bindingfragment thereof, an aptamer, an inhibitory nucleic acid, and aninhibitory polypeptide. In certain embodiments that may be combined withany of the preceding embodiments, the antagonist of TIGIT expressionand/or activity is an anti-TIGIT antibody or antigen-binding fragmentthereof. In certain embodiments that may be combined with any of thepreceding embodiments, the antagonist of TIGIT expression and/oractivity is an inhibitory nucleic acid selected from the groupconsisting of an antisense polynucleotide, an interfering RNA, acatalytic RNA, and an RNA-DNA chimera. In certain embodiments that maybe combined with any of the preceding embodiments, the antagonist of PVRexpression and/or activity is selected from the group consisting of asmall molecule inhibitor, an inhibitory antibody or antigen-bindingfragment thereof, an aptamer, an inhibitory nucleic acid, and aninhibitory polypeptide. In certain embodiments that may be combined withany of the preceding embodiments, the agent that inhibits and/or blocksthe interaction of TIGIT with PVR is selected from the group consistingof a small molecule inhibitor, an inhibitory antibody or antigen-bindingfragment thereof, an aptamer, an inhibitory nucleic acid, and aninhibitory polypeptide. In certain embodiments that may be combined withany of the preceding embodiments, the agent that inhibits and/or blocksthe interaction of TIGIT with PVRL2 is selected from the groupconsisting of a small molecule inhibitor, an inhibitory antibody orantigen-binding fragment thereof, an aptamer, an inhibitory nucleicacid, and an inhibitory polypeptide. In certain embodiments that may becombined with any of the preceding embodiments, the agent that inhibitsand/or blocks the interaction of TIGIT with PVRL3 is selected from thegroup consisting of a small molecule inhibitor, an inhibitory antibodyor antigen-binding fragment thereof, an aptamer, an inhibitory nucleicacid, and an inhibitory polypeptide. In certain embodiments that may becombined with any of the preceding embodiments, the agent that inhibitsand/or blocks the intracellular signaling mediated by TIGIT binding toPVR is selected from the group consisting of a small molecule inhibitor,an inhibitory antibody or antigen-binding fragment thereof, an aptamer,an inhibitory nucleic acid, and an inhibitory polypeptide. In certainembodiments that may be combined with any of the preceding embodiments,the agent that inhibits and/or blocks the interaction of TIGIT withPVRL2 is selected from the group consisting of a small moleculeinhibitor, an inhibitory antibody or antigen-binding fragment thereof,an aptamer, an inhibitory nucleic acid, and an inhibitory polypeptide.In certain embodiments that may be combined with any of the precedingembodiments, the agent that inhibits and/or blocks the interaction ofTIGIT with PVRL3 is selected from the group consisting of a smallmolecule inhibitor, an inhibitory antibody or antigen-binding fragmentthereof, an aptamer, an inhibitory nucleic acid, and an inhibitorypolypeptide.

In other aspects, the present disclosure provides a method ofincreasing, enhancing, or stimulating an immune response or function inan individual comprising administering to the individual an effectiveamount of an agent that decreases or inhibits TIGIT expression and/oractivity and an agent that decreases or inhibits one or more additionalimmune co-inhibitory receptors. In other aspects, the present disclosureprovides use of an effective amount of an agent that decreases orinhibits TIGIT expression and/or activity in the manufacture of amedicament for enhancing or stimulating an immune response or functionin an individual, wherein the agent that decreases or inhibits TIGITexpression and/or activity is used in combination with an agent thatdecreases or inhibits one or more additional immune co-inhibitoryreceptors. In other aspects, the present disclosure provides use of aneffective amount of an agent that decreases or inhibits one or moreadditional immune co-inhibitory receptors in the manufacture of amedicament for enhancing or stimulating an immune response or functionin an individual, wherein the agent that decreases or inhibits one ormore additional immune co-inhibitory receptors is used in combinationwith an agent that decreases or inhibits TIGIT expression and/oractivity. In other aspects, the present disclosure provides apharmaceutical composition comprising an agent that decreases orinhibits TIGIT expression and/or activity for use in enhancing orstimulating an immune response or function in combination with an agentthat decreases or inhibits one or more additional immune co-inhibitoryreceptors. In other aspects, the present disclosure provides apharmaceutical composition comprising an agent that decreases orinhibits one or more additional immune co-inhibitory receptors for usein enhancing or stimulating an immune response or function incombination with an agent that decreases or inhibits TIGIT expressionand/or activity. In other aspects, the present disclosure provides acombination comprising an effective amount of an agent that decreases orinhibits TIGIT expression and/or activity and an agent that decreases orinhibits one or more additional immune co-inhibitory receptors. Incertain embodiments that may be combined with any of the precedingembodiments, the one or more additional immune co-inhibitory receptor isselected from the group consisting of PD-1, CTLA-4, LAG3, TIM3, BTLA,VISTA, B7H4, and CD96. In certain embodiments that may be combined withany of the preceding embodiments, the one or more additional immuneco-inhibitory receptor is selected from the group consisting of PD-1,CTLA-4, LAG3 and TIM3.

In other aspects, the present disclosure provides a method ofincreasing, enhancing, or stimulating an immune response or function inan individual comprising administering to the individual an effectiveamount of an agent that decreases or inhibits TIGIT expression and/oractivity and an agent that increases or activates one or more additionalimmune co-stimulatory receptors. In other aspects, the presentdisclosure provides use of an effective amount of an agent thatdecreases or inhibits TIGIT expression and/or activity in themanufacture of a medicament for enhancing or stimulating an immuneresponse or function in an individual, wherein the agent that decreasesor inhibits TIGIT expression and/or activity is used in combination withan agent that increases or activates one or more additional immuneco-stimulatory receptors. In other aspects, the present disclosureprovides use of an effective amount of an a agent that increases oractivates one or more additional immune co-stimulatory receptors in themanufacture of a medicament for enhancing or stimulating an immuneresponse or function in an individual, wherein the a agent thatincreases or activates one or more additional immune co-stimulatoryreceptors is used in combination with an agent that decreases orinhibits TIGIT expression and/or activity. In other aspects, the presentdisclosure provides a pharmaceutical composition comprising an agentthat decreases or inhibits TIGIT expression and/or activity for use inenhancing or stimulating an immune response or function in combinationwith an agent that increases or activates one or more additional immuneco-stimulatory receptors. In other aspects, the present disclosureprovides a pharmaceutical composition comprising an agent that increasesor activates one or more additional immune co-stimulatory receptors foruse in enhancing or stimulating an immune response or function incombination with an agent that decreases or inhibits TIGIT expressionand/or activity. In other aspects, the present disclosure provides acombination comprising an effective amount of an agent that decreases orinhibits TIGIT expression and/or activity and an agent that increases oractivates one or more additional immune co-stimulatory receptors. Incertain embodiments that may be combined with any of the precedingembodiments, the one or more additional immune co-stimulatory receptorsis selected from the group consisting of CD226, OX-40, CD28, CD27,CD137, HVEM, GITR, MICA, ICOS, NKG2D, and 2B4. In certain embodimentsthat may be combined with any of the preceding embodiments, the one ormore additional immune co-stimulatory receptors is selected from thegroup consisting of CD226, OX-40, CD27, CD137, HVEM and GITR. In certainembodiments that may be combined with any of the preceding embodiments,the one or more additional immune co-stimulatory receptors is selectedfrom the group consisting of OX-40 and CD27.

In certain embodiments that may be combined with any of the precedingembodiments, the method further comprises administering at least onechemotherapeutic agent. In certain embodiments that may be combined withany of the preceding embodiments, the individual has cancer. In certainembodiments that may be combined with any of the preceding embodiments,the individual is a human. In certain embodiments that may be combinedwith any of the preceding embodiments, CD4 and/or CD8 T cells in theindividual have increased or enhanced priming, activation,proliferation, cytokine release and/or cytolytic activity relative toprior to the administration of the combination. In certain embodimentsthat may be combined with any of the preceding embodiments, the numberof CD4 and/or CD8 T cells is elevated relative to prior toadministration of the combination. In certain embodiments that may becombined with any of the preceding embodiments, the number of activatedCD4 and/or CD8 T cells is elevated relative to prior to administrationof the combination. In certain embodiments that may be combined with anyof the preceding embodiments, activated CD4 and/or CD8 T cells arecharacterized by γ-IFN⁺ producing CD4 and/or CD8 T cells and/or enhancedcytolytic activity relative to prior to the administration of thecombination. In certain embodiments that may be combined with any of thepreceding embodiments, the CD4 and/or CD8 T cells exhibit increasedrelease of cytokines selected from the group consisting of IFN-γ, TNF-α,and interleukins. In certain embodiments that may be combined with anyof the preceding embodiments, the CD4 and/or CD8 T cells are effectormemory T cells. In certain embodiments that may be combined with any ofthe preceding embodiments, the CD4 and/or CD8 effector memory T cellsare characterized by γ-IFN⁺ producing CD4 and/or CD8 T cells and/orenhanced cytolytic activity. In certain embodiments that may be combinedwith any of the preceding embodiments, the CD4 and/or CD8 effectormemory T cells are characterized by having the expression of CD44^(high)CD62L^(low). In certain embodiments that may be combined with any of thepreceding embodiments, the cancer has elevated levels of T cellinfiltration. In certain embodiments that may be combined with any ofthe preceding embodiments, the agent that decreases or inhibits TIGITexpression and/or activity is selected from the group consisting of anantagonist of TIGIT expression and/or activity, an antagonist of PVRexpression and/or activity, an agent that inhibits and/or blocks theinteraction of TIGIT with PVR, an agent that inhibits and/or blocks theinteraction of TIGIT with PVRL2, an agent that inhibits and/or blocksthe interaction of TIGIT with PVRL3, an agent that inhibits and/orblocks the intracellular signaling mediated by TIGIT binding to PVR, anagent that inhibits and/or blocks the intracellular signaling mediatedby TIGIT binding to PVRL2, an agent that inhibits and/or blocks theintracellular signaling mediated by TIGIT binding to PVRL3, andcombinations thereof. In certain embodiments that may be combined withany of the preceding embodiments, the antagonist of TIGIT expressionand/or activity is selected from the group consisting of a smallmolecule inhibitor, an inhibitory antibody or antigen-binding fragmentthereof, an aptamer, an inhibitory nucleic acid, and an inhibitorypolypeptide. In certain embodiments that may be combined with any of thepreceding embodiments, the antagonist of PVR expression and/or activityis selected from the group consisting of a small molecule inhibitor, aninhibitory antibody or antigen-binding fragment thereof, an aptamer, aninhibitory nucleic acid, and an inhibitory polypeptide. In certainembodiments that may be combined with any of the preceding embodiments,the agent that inhibits and/or blocks the interaction of TIGIT with PVRis selected from the group consisting of a small molecule inhibitor, aninhibitory antibody or antigen-binding fragment thereof, an aptamer, aninhibitory nucleic acid, and an inhibitory polypeptide. In certainembodiments that may be combined with any of the preceding embodiments,the agent that inhibits and/or blocks the interaction of TIGIT withPVRL2 is selected from the group consisting of a small moleculeinhibitor, an inhibitory antibody or antigen-binding fragment thereof,an aptamer, an inhibitory nucleic acid, and an inhibitory polypeptide.In certain embodiments that may be combined with any of the precedingembodiments, the agent that inhibits and/or blocks the interaction ofTIGIT with PVRL3 is selected from the group consisting of a smallmolecule inhibitor, an inhibitory antibody or antigen-binding fragmentthereof, an aptamer, an inhibitory nucleic acid, and an inhibitorypolypeptide. In certain embodiments that may be combined with any of thepreceding embodiments, the agent that inhibits and/or blocks theintracellular signaling mediated by TIGIT binding to PVR is selectedfrom the group consisting of a small molecule inhibitor, an inhibitoryantibody or antigen-binding fragment thereof, an aptamer, an inhibitorynucleic acid, and an inhibitory polypeptide. In certain embodiments thatmay be combined with any of the preceding embodiments, the agent thatinhibits and/or blocks the intracellular signaling mediated by TIGITbinding to PVRL2 is selected from the group consisting of a smallmolecule inhibitor, an inhibitory antibody or antigen-binding fragmentthereof, an aptamer, an inhibitory nucleic acid, and an inhibitorypolypeptide. In certain embodiments that may be combined with any of thepreceding embodiments, the agent that inhibits and/or blocks theintracellular signaling mediated by TIGIT binding to PVRL3 is selectedfrom the group consisting of a small molecule inhibitor, an inhibitoryantibody or antigen-binding fragment thereof, an aptamer, an inhibitorynucleic acid, and an inhibitory polypeptide. In certain embodiments thatmay be combined with any of the preceding embodiments, the antagonist ofTIGIT expression and/or activity is an inhibitory nucleic acid selectedfrom the group consisting of an antisense polynucleotide, an interferingRNA, a catalytic RNA, and an RNA-DNA chimera. In certain embodimentsthat may be combined with any of the preceding embodiments, theantisense polynucleotide targets TIGIT.

In certain embodiments that may be combined with any of the precedingembodiments, the interfering RNA targets TIGIT. In certain embodimentsthat may be combined with any of the preceding embodiments, thecatalytic RNA targets TIGIT. In certain embodiments that may be combinedwith any of the preceding embodiments, the RNA-DNA chimera targetsTIGIT. In certain embodiments that may be combined with any of thepreceding embodiments, the antagonist of TIGIT expression and/oractivity is an anti-TIGIT antibody or antigen-binding fragment thereof.In certain embodiments that may be combined with any of the precedingembodiments, the anti-TIGIT antibody or antigen-binding fragment thereofcomprises at least one HVR comprising an amino acid sequence selectedfrom the amino acid sequences (1) KSSQSLYYSGVKENLLA (SEQ ID NO:1),ASIRFT (SEQ ID NO:2), QQGINNPLT (SEQ ID NO:3), GFTFSSFTMH (SEQ ID NO:4),FIRSGSGIVFYADAVRG (SEQ ID NO:5), and RPLGHNTFDS (SEQ ID NO:6); or (2)RSSQSLVNSYGNTFLS (SEQ ID NO:7), GISNRFS (SEQ ID NO:8), LQGTHQPPT (SEQ IDNO:9), GYSFTGHLMN (SEQ ID NO:10), LIIPYNGGTSYNQKFKG (SEQ ID NO:11), andGLRGFYAMDY (SEQ ID NO:12). In certain embodiments that may be combinedwith any of the preceding embodiments, the anti-TIGIT antibody orantigen-binding fragment thereof, wherein the antibody light chaincomprises the amino acid sequence set forth inDIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGT KLEIKR (SEQID NO:13) orDVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPPTFGPGTKLEVK (SEQ ID NO:14). Incertain embodiments that may be combined with any of the precedingembodiments, the anti-TIGIT antibody or antigen-binding fragmentthereof, wherein the antibody heavy chain comprises the amino acidsequence set forth inEVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLV TVSS (SEQ IDNO:15) or EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGLIIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGLRGFYAMDYWGQGTSV TVSS (SEQ IDNO:16). In certain embodiments that may be combined with any of thepreceding embodiments, the anti-TIGIT antibody or antigen-bindingfragment thereof, wherein the antibody light chain comprises the aminoacid sequence set forth inDIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGT KLEIKR (SEQID NO:13) orDVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPPTFGPGTKLEVK (SEQ ID NO:14), andthe antibody heavy chain comprises the amino acid sequence set forth inEVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLV TVSS (SEQ IDNO:15) or EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGLIIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGLRGFYAMDYWGQGTSV TVSS (SEQ IDNO: 16). In certain embodiments that may be combined with any of thepreceding embodiments, the anti-TIGIT antibody or antigen-bindingfragment thereof, wherein the antibody is selected from the groupconsisting of a humanized antibody, a chimeric antibody, a bispecificantibody, a heteroconjugate antibody, and an immunotoxin. In certainembodiments that may be combined with any of the preceding embodiments,the anti-TIGIT antibody or antigen-binding fragment thereof comprises atleast one HVR that is at least 90% identical to an HVR set forth in anyone of (1) KSSQSLYYSGVKENLLA (SEQ ID NO:1), ASIRFT (SEQ ID NO:2),QQGINNPLT (SEQ ID NO:3), GFTFSSFTMH (SEQ ID NO:4), FIRSGSGIVFYADAVRG(SEQ ID NO:5), and RPLGHNTFDS (SEQ ID NO:6); or (2) RSSQSLVNSYGNTFLS(SEQ ID NO:7), GISNRFS (SEQ ID NO:8), LQGTHQPPT (SEQ ID NO:9),GYSFTGHLMN (SEQ ID NO:10), LIIPYNGGTSYNQKFKG (SEQ ID NO:11), andGLRGFYAMDY (SEQ ID NO:12). In certain embodiments that may be combinedwith any of the preceding embodiments, the anti-TIGIT antibody orfragment thereof comprises the light chain comprising amino acidsequences at least 90% identical to the amino acid sequences set forthin DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGT KLEIKR (SEQID NO:13) orDVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPPTFGPGTKLEVK (SEQ ID NO:14);and/or the heavy chain comprising amino acid sequences at least 90%identical to the amino acid sequences set forth inEVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLV TVSS (SEQ IDNO:15) or EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGLIIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGLRGFYAMDYWGQGTSV TVSS (SEQ IDNO:16). In certain embodiments that may be combined with any of thepreceding embodiments, the PD-1 axis binding antagonist is selected fromthe group consisting of a PD-1 binding antagonist, a PD-L1 bindingantagonist and a PD-L2 binding antagonist. In certain embodiments thatmay be combined with any of the preceding embodiments, the PD-1 axisbinding antagonist is a PD-1 binding antagonist. In certain embodimentsthat may be combined with any of the preceding embodiments, the PD-1binding antagonist inhibits the binding of PD-1 to its ligand bindingpartners. In certain embodiments that may be combined with any of thepreceding embodiments, the PD-1 binding antagonist inhibits the bindingof PD-1 to PD-L1. In certain embodiments that may be combined with anyof the preceding embodiments, the PD-1 binding antagonist inhibits thebinding of PD-1 to PD-L2. In certain embodiments that may be combinedwith any of the preceding embodiments, the PD-1 binding antagonistinhibits the binding of PD-1 to both PD-L1 and PD-L2. In certainembodiments that may be combined with any of the preceding embodiments,the PD-1 binding antagonist is an antibody. In certain embodiments thatmay be combined with any of the preceding embodiments, the PD-1 bindingantagonist is MDX-1106. In certain embodiments that may be combined withany of the preceding embodiments, the PD-1 binding antagonist isMK-3475. In certain embodiments that may be combined with any of thepreceding embodiments, the PD-1 binding antagonist is CT-011. In certainembodiments that may be combined with any of the preceding embodiments,the PD-1 binding antagonist is AMP-224. In certain embodiments that maybe combined with any of the preceding embodiments, the PD-1 axis bindingantagonist is a PD-L1 binding antagonist. In certain embodiments thatmay be combined with any of the preceding embodiments, the PD-L1 bindingantagonist inhibits the binding of PD-L1 to PD-1. In certain embodimentsthat may be combined with any of the preceding embodiments, the PD-L1binding antagonist inhibits the binding of PD-L1 to B7-1. In certainembodiments that may be combined with any of the preceding embodiments,the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1and B7-1. In certain embodiments that may be combined with any of thepreceding embodiments, the PD-L1 binding antagonist is an anti-PD-L1antibody. In certain embodiments that may be combined with any of thepreceding embodiments, the PD-L1 binding antagonist is selected from thegroup consisting of YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736. Incertain embodiments that may be combined with any of the precedingembodiments, the anti-PD-L1antibody comprises a heavy chain comprisingHVR-H1 sequence of GFTFSDSWIH (SEQ ID NO:17), HVR-H2 sequence ofAWISPYGGSTYYADSVKG (SEQ ID NO:18), and HVR-H3 sequence of RHWPGGFDY (SEQID NO:19); and a light chain comprising HVR-L1 sequence of RASQDVSTAVA(SEQ ID NO:20), HVR-L2 sequence of SASFLYS (SEQ ID NO:21), and HVR-L3sequence of QQYLYHPAT (SEQ ID NO:22). In certain embodiments that may becombined with any of the preceding embodiments, the anti-PD-L1antibodycomprises a heavy chain variable region comprising the amino acidsequence of EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVT VSA (SEQ IDNO:23), EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVT VSSASTK (SEQID NO:40), or EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVT VSS (SEQ IDNO:41), and a light chain variable region comprising the amino acidsequence of DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO:24). Incertain embodiments that may be combined with any of the precedingembodiments, the PD-1 axis binding antagonist is a PD-L2 bindingantagonist. In certain embodiments that may be combined with any of thepreceding embodiments, the PD-L2 binding antagonist is an antibody. Incertain embodiments that may be combined with any of the precedingembodiments, the PD-L2 binding antagonist is an immunoadhesin. Incertain embodiments that may be combined with any of the precedingembodiments, the cancer is selected from the group consisting ofnon-small cell lung cancer, small cell lung cancer, renal cell cancer,colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer,gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma,melanoma, head and neck cancer, thyroid cancer, sarcoma, prostatecancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia,lymphomas, myelomas, mycoses fungoids, merkel cell cancer, and otherhematologic malignancies. In certain embodiments that may be combinedwith any of the preceding embodiments, the agent that decreases orinhibits TIGIT expression and/or activity is administered continuously.In certain embodiments that may be combined with any of the precedingembodiments, the agent that decreases or inhibits TIGIT expressionand/or activity is administered intermittently. In certain embodimentsthat may be combined with any of the preceding embodiments, the agentthat decreases or inhibits TIGIT expression and/or activity isadministered before the PD-1 axis binding antagonist. In certainembodiments that may be combined with any of the preceding embodiments,the agent that decreases or inhibits TIGIT expression and/or activity isadministered simultaneous with the PD-1 axis binding antagonist. Incertain embodiments that may be combined with any of the precedingembodiments, the agent that decreases or inhibits TIGIT expressionand/or activity is administered after the PD-1 axis binding antagonist.In certain embodiments that may be combined with any of the precedingembodiments, the PD-1 axis binding antagonist is administered before theagent that modulates CD226 expression and/or activity. In certainembodiments that may be combined with any of the preceding embodiments,the PD-1 axis binding antagonist is administered simultaneous with theagent that modulates CD226 expression and/or activity. In certainembodiments that may be combined with any of the preceding embodiments,the PD-1 axis binding antagonist is administered after the agent thatmodulates CD226 expression and/or activity. In certain embodiments thatmay be combined with any of the preceding embodiments, the agent thatdecreases or inhibits TIGIT expression and/or activity is administeredbefore the agent that decreases or inhibits one or more additionalimmune co-inhibitory receptors. In certain embodiments that may becombined with any of the preceding embodiments, the agent that decreasesor inhibits TIGIT expression and/or activity is administeredsimultaneous with the agent that decreases or inhibits one or moreadditional immune co-inhibitory receptors. In certain embodiments thatmay be combined with any of the preceding embodiments, the agent thatdecreases or inhibits TIGIT expression and/or activity is administeredafter the agent that decreases or inhibits one or more additional immuneco-inhibitory receptors. In certain embodiments that may be combinedwith any of the preceding embodiments, the agent that decreases orinhibits TIGIT expression and/or activity is administered before theagent that increases or activates one or more additional immuneco-stimulatory receptors. In certain embodiments that may be combinedwith any of the preceding embodiments, the agent that decreases orinhibits TIGIT expression and/or activity is administered simultaneouswith the agent that increases or activates one or more additional immuneco-stimulatory receptors. In certain embodiments that may be combinedwith any of the preceding embodiments, the agent that decreases orinhibits TIGIT expression and/or activity is administered after theagent that increases or activates one or more additional immuneco-stimulatory receptors.

In other aspects, the present disclosure provides a kit comprising aPD-1 axis binding antagonist and a package insert comprisinginstructions for using the PD-1 axis binding antagonist in combinationwith an agent that decreases or inhibits TIGIT expression and/oractivity to treat or delay progression of cancer in an individual.

In other aspects, the present disclosure provides a kit comprising aPD-1 axis binding antagonist and an agent that decreases or inhibitsTIGIT expression and/or activity, and a package insert comprisinginstructions for using the PD-1 axis binding antagonist and the agentthat decreases or inhibits TIGIT expression and/or activity to treat ordelay progression of cancer in an individual.

In other aspects, the present disclosure provides a kit comprising anagent that decreases or inhibits TIGIT expression and/or activity and apackage insert comprising instructions for using the agent thatdecreases or inhibits TIGIT expression and/or activity in combinationwith a PD-1 axis binding antagonist to treat or delay progression ofcancer in an individual.

In other aspects, the present disclosure provides a kit comprising aPD-1 axis binding antagonist and a package insert comprisinginstructions for using the PD-1 axis binding antagonist in combinationwith an agent that decreases or inhibits TIGIT expression and/oractivity to enhance immune function of an individual having cancer.

In other aspects, the present disclosure provides a kit comprising aPD-1 axis binding antagonist and an agent that decreases or inhibitsTIGIT expression and/or activity, and a package insert comprisinginstructions for using the PD-1 axis binding antagonist and the agentthat decreases or inhibits TIGIT expression and/or activity to enhanceimmune function of an individual having cancer.

In other aspects, the present disclosure provides a kit comprising anagent that decreases or inhibits TIGIT expression and/or activity and apackage insert comprising instructions for using the agent thatdecreases or inhibits TIGIT expression and/or activity in combinationwith a PD-1 axis binding antagonist to enhance immune function of anindividual having cancer.

In other aspects, the present disclosure provides a kit comprising aPD-1 axis binding antagonist and a package insert comprisinginstructions for using the PD-1 axis binding antagonist in combinationwith an agent that modulates CD226 expression and/or activity to treator delay progression of cancer in an individual.

In other aspects, the present disclosure provides a kit comprising aPD-1 axis binding antagonist and an agent that modulates CD226expression and/or activity, and a package insert comprising instructionsfor using the PD-1 axis binding antagonist and the agent that modulatesCD226 expression and/or activity to treat or delay progression of cancerin an individual.

In other aspects, the present disclosure provides a kit comprising anagent that modulates CD226 expression and/or activity and a packageinsert comprising instructions for using the agent modulates CD226expression and/or activity in combination with a PD-1 axis bindingantagonist to treat or delay progression of cancer in an individual.

In other aspects, the present disclosure provides a kit comprising aPD-1 axis binding antagonist and a package insert comprisinginstructions for using the PD-1 axis binding antagonist in combinationwith an agent that modulates CD226 expression and/or activity to enhanceimmune function of an individual having cancer.

In other aspects, the present disclosure provides a kit comprising aPD-1 axis binding antagonist and an agent that modulates CD226expression and/or activity, and a package insert comprising instructionsfor using the PD-1 axis binding antagonist and the agent that modulatesCD226 expression and/or activity to enhance immune function of anindividual having cancer.

In other aspects, the present disclosure provides a kit comprising anagent modulates CD226 expression and/or activity and a package insertcomprising instructions for using the agent that modulates CD226expression and/or activity in combination with a PD-1 axis bindingantagonist to enhance immune function of an individual having cancer.

In certain embodiments that may be combined with any of the precedingembodiments, the PD-1 axis binding antagonist is an anti-PD-L1 antibody.In certain embodiments that may be combined with any of the precedingembodiments, the anti-PD-L1 antibody is selected from the groupconsisting of YW243.55.S70, MPDL3280A, MDX-1105 and MEDI4736. In certainembodiments that may be combined with any of the preceding embodiments,the anti-PD-L1antibody comprises a heavy chain comprising HVR-H1sequence of GFTFSDSWIH (SEQ ID NO:17), HVR-H2 sequence ofAWISPYGGSTYYADSVKG (SEQ ID NO:18), and HVR-H3 sequence of RHWPGGFDY (SEQID NO:19); and a light chain comprising HVR-L1 sequence of RASQDVSTAVA(SEQ ID NO:20), HVR-L2 sequence of SASFLYS (SEQ ID NO:21), and HVR-L3sequence of QQYLYHPAT (SEQ ID NO:22). In certain embodiments that may becombined with any of the preceding embodiments, the anti-PD-L1antibodycomprises a heavy chain variable region comprising the amino acidsequence of EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVT VSA (SEQ IDNO:23), EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVT VSSASTK (SEQID NO:40), or EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVT VSS (SEQ IDNO:41), and a light chain variable region comprising the amino acidsequence of DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO:24). Incertain embodiments that may be combined with any of the precedingembodiments, the PD-1 axis binding antagonist is an anti-PD-1 antibody.In certain embodiments that may be combined with any of the precedingembodiments, the anti-PD-1 antibody is MDX-1106, MK-3475, or CT-011. Incertain embodiments that may be combined with any of the precedingembodiments, the PD-1 axis binding antagonist is AMP-224. In certainembodiments that may be combined with any of the preceding embodiments,the PD-1 axis binding antagonist is a PD-L2 binding antagonist. Incertain embodiments that may be combined with any of the precedingembodiments, the PD-L2 binding antagonist is an antibody. In certainembodiments that may be combined with any of the preceding embodiments,the PD-L2 binding antagonist is an immunoadhesin.

In other aspects, the present disclosure provides a kit comprising anagent that decreases or inhibits TIGIT expression and/or activity and apackage insert comprising instructions for using the agent thatdecreases or inhibits TIGIT expression and/or activity in combinationwith an agent that decreases or inhibits one or more additional immuneco-inhibitory receptors to treat or delay progression of cancer in anindividual. In other aspects, the present disclosure provides a kitcomprising an agent that decreases or inhibits TIGIT expression and/oractivity and an agent that decreases or inhibits one or more additionalimmune co-inhibitory receptors, and a package insert comprisinginstructions for using the agent that decreases or inhibits TIGITexpression and/or activity and the agent that decreases or inhibits oneor more additional immune co-inhibitory receptors to treat or delayprogression of cancer in an individual. In other aspects, the presentdisclosure provides a kit comprising an agent that decreases or inhibitsone or more additional immune co-inhibitory receptors and a packageinsert comprising instructions for using the agent that decreases orinhibits one or more additional immune co-inhibitory receptors incombination with an agent that decreases or inhibits TIGIT expressionand/or activity to treat or delay progression of cancer in anindividual. In other aspects, the present disclosure provides a kitcomprising an agent that decreases or inhibits TIGIT expression and/oractivity and a package insert comprising instructions for using theagent that decreases or inhibits TIGIT expression and/or activity incombination with an agent that decreases or inhibits one or moreadditional immune co-inhibitory receptors to enhance immune function ofan individual having cancer. In other aspects, the present disclosureprovides a kit comprising an agent that decreases or inhibits TIGITexpression and/or activity and an agent that decreases or inhibits oneor more additional immune co-inhibitory receptors, and a package insertcomprising instructions for using the agent that decreases or inhibitsTIGIT expression and/or activity and the agent that decreases orinhibits one or more additional immune co-inhibitory receptors toenhance immune function of an individual having cancer. In otheraspects, the present disclosure provides a kit comprising an agent thatdecreases or inhibits one or more additional immune co-inhibitoryreceptors and a package insert comprising instructions for using theagent that decreases or inhibits one or more additional immuneco-inhibitory receptors in combination with an agent that decreases orinhibits TIGIT expression and/or activity to enhance immune function ofan individual having cancer. In certain embodiments that may be combinedwith any of the preceding embodiments, the one or more additional immuneco-inhibitory receptor is selected from the group consisting of PD-1,CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, and CD96. In certain embodimentsthat may be combined with any of the preceding embodiments, the one ormore additional immune co-inhibitory receptor is selected from the groupconsisting of PD-1, CTLA-4, LAG3 and TIM3.

In other aspects, the present disclosure provides a kit comprising anagent that decreases or inhibits TIGIT expression and/or activity and apackage insert comprising instructions for using the agent thatdecreases or inhibits TIGIT expression and/or activity in combinationwith an agent that increases or activates one or more additional immuneco-stimulatory receptors to treat or delay progression of cancer in anindividual. In other aspects, the present disclosure provides a kitcomprising an agent that decreases or inhibits TIGIT expression and/oractivity and an agent that increases or activates one or more additionalimmune co-stimulatory receptors, and a package insert comprisinginstructions for using the agent that decreases or inhibits TIGITexpression and/or activity and the agent that increases or activates oneor more additional immune co-stimulatory receptors to treat or delayprogression of cancer in an individual. In other aspects, the presentdisclosure provides a kit comprising an agent that increases oractivates one or more additional immune co-stimulatory receptors and apackage insert comprising instructions for using the agent thatincreases or activates one or more additional immune co-stimulatoryreceptors in combination with an agent that decreases or inhibits TIGITexpression and/or activity to treat or delay progression of cancer in anindividual. In other aspects, the present disclosure provides a kitcomprising an agent that decreases or inhibits TIGIT expression and/oractivity and a package insert comprising instructions for using theagent that decreases or inhibits TIGIT expression and/or activity incombination with an agent that increases or activates one or moreadditional immune co-stimulatory receptors to enhance immune function ofan individual having cancer. In other aspects, the present disclosureprovides a kit comprising an agent that decreases or inhibits TIGITexpression and/or activity and an agent that increases or activates oneor more additional immune co-stimulatory receptors, and a package insertcomprising instructions for using the agent that decreases or inhibitsTIGIT expression and/or activity and the agent that increases oractivates one or more additional immune co-stimulatory receptors toenhance immune function of an individual having cancer. In otheraspects, the present disclosure provides a kit comprising an agent thatincreases or activates one or more additional immune co-stimulatoryreceptors and a package insert comprising instructions for using theagent that increases or activates one or more additional immuneco-stimulatory receptors in combination with an agent that decreases orinhibits TIGIT expression and/or activity to enhance immune function ofan individual having cancer. In certain embodiments that may be combinedwith any of the preceding embodiments, the or more additional immuneco-stimulatory receptor is selected from the group consisting of CD226,OX-40, CD28, CD27, CD137, HVEM, GITR, MICA, ICOS, NKG2D, and 2B4. Incertain embodiments that may be combined with any of the precedingembodiments, the one or more additional immune co-stimulatory receptoris selected from the group consisting of CD226, OX-40, CD27, CD137, HVEMand GITR. In certain embodiments that may be combined with any of thepreceding embodiments, the one or more additional immune co-stimulatoryreceptor is selected from the group consisting of OX-40 and CD27.

In certain embodiments that may be combined with any of the precedingembodiments, the individual is a human. In certain embodiments that maybe combined with any of the preceding embodiments, the agent thatdecreases or inhibits TIGIT expression and/or activity is selected fromthe group consisting of an antagonist of TIGIT expression and/oractivity, an antagonist of PVR expression and/or activity, an agent thatinhibits and/or blocks the interaction of TIGIT with PVR, an agent thatinhibits and/or blocks the interaction of TIGIT with PVRL2, an agentthat inhibits and/or blocks the interaction of TIGIT with PVRL3, anagent that inhibits and/or blocks the intracellular signaling mediatedby TIGIT binding to PVR, an agent that inhibits and/or blocks theintracellular signaling mediated by TIGIT binding to PVRL2, and an agentthat inhibits and/or blocks the intracellular signaling mediated byTIGIT binding to PVRL3. In certain embodiments that may be combined withany of the preceding embodiments, the antagonist of TIGIT expressionand/or activity is an anti-TIGIT antibody or antigen-binding fragmentthereof. In certain embodiments that may be combined with any of thepreceding embodiments, the agent that modulates CD226 expression and/oractivity is an agent that increases and/or stimulates CD226 expressionand/or activity. In certain embodiments that may be combined with any ofthe preceding embodiments, the agent that modulates CD226 expressionand/or activity is an agent that increases and/or stimulates theinteraction of CD226 with PVR. In certain embodiments that may becombined with any of the preceding embodiments, the agent that modulatesCD226 expression and/or activity is an agent that increases and/orstimulates the intracellular signaling mediated by CD226 binding to PVR.In certain embodiments that may be combined with any of the precedingembodiments, the agent that modulates CD226 expression and/or activityis selected from the group consisting of an agent that inhibits and/orblocks the interaction of CD226 with TIGIT, an antagonist of TIGITexpression and/or activity, an antagonist of PVR expression and/oractivity, an agent that inhibits and/or blocks the interaction of TIGITwith PVR, an agent that inhibits and/or blocks the interaction of TIGITwith PVRL2, an agent that inhibits and/or blocks the interaction ofTIGIT with PVRL3, an agent that inhibits and/or blocks the intracellularsignaling mediated by TIGIT binding to PVR, an agent that inhibitsand/or blocks the intracellular signaling mediated by TIGIT binding toPVRL2, and an agent that inhibits and/or blocks the intracellularsignaling mediated by TIGIT binding to PVRL3. In certain embodimentsthat may be combined with any of the preceding embodiments, the agentthat modulates CD226 expression and/or activity is an agent thatinhibits and/or blocks the interaction of CD226 with TIGIT. In certainembodiments that may be combined with any of the preceding embodiments,the agent that inhibits and/or blocks the interaction of CD226 withTIGIT is a small molecule inhibitor, an inhibitory antibody orantigen-binding fragment thereof, an aptamer, an inhibitory nucleicacid, or an inhibitory polypeptide. In certain embodiments that may becombined with any of the preceding embodiments, the agent that inhibitsand/or blocks the interaction of CD226 with TIGIT is an anti-TIGITantibody or antigen-binding fragment thereof. In certain embodimentsthat may be combined with any of the preceding embodiments, theanti-TIGIT antibody or antigen-binding fragment thereof comprises atleast one HVR comprising an amino acid sequence selected from the aminoacid sequences (1) KSSQSLYYSGVKENLLA (SEQ ID NO:1), ASIRFT (SEQ IDNO:2), QQGINNPLT (SEQ ID NO:3), GFTFSSFTMH (SEQ ID NO:4),FIRSGSGIVFYADAVRG (SEQ ID NO:5), and RPLGHNTFDS (SEQ ID NO:6); or (2)RSSQSLVNSYGNTFLS (SEQ ID NO:7), GISNRFS (SEQ ID NO:8), LQGTHQPPT (SEQ IDNO:9), GYSFTGHLMN (SEQ ID NO:10), LIIPYNGGTSYNQKFKG (SEQ ID NO:11), andGLRGFYAMDY (SEQ ID NO:12). In certain embodiments that may be combinedwith any of the preceding embodiments, the anti-TIGIT antibody orantigen-binding fragment thereof, wherein the antibody light chaincomprises the amino acid sequence set forth inDIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGT KLEIKR (SEQID NO:13) orDVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPPTFGPGTKLEVK (SEQ ID NO:14). Incertain embodiments that may be combined with any of the precedingembodiments, the anti-TIGIT antibody or antigen-binding fragmentthereof, wherein the antibody heavy chain comprises the amino acidsequence set forth inEVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLV TVSS (SEQ IDNO:15) or EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGLIIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGLRGFYAMDYWGQGTSV TVSS (SEQ IDNO:16). In certain embodiments that may be combined with any of thepreceding embodiments, the anti-TIGIT antibody or antigen-bindingfragment thereof, wherein the antibody light chain comprises the aminoacid sequence set forth inDIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGT KLEIKR (SEQID NO:13) orDVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPPTFGPGTKLEVK (SEQ ID NO:14), andthe antibody heavy chain comprises the amino acid sequence set forth inEVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLV TVSS (SEQ IDNO:15) or EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGLIIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGLRGFYAMDYWGQGTSV TVSS (SEQ IDNO: 16).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1F show that TIGIT is highly expressed on exhausted CD8+ andCD4+ T cells. FIG. 1A-1D depict MACS-enriched C57BL6/J splenic CD8+ Tcells that were stimulated with plate-bound anti-CD3 and anti-CD28 for24-48 hours in vitro. Flow cytometry histograms representative of TIGITexpression (red) relative to isotype staining (gray). Quantitation ofTIGIT MFI is also shown. ***, P<0.001. Data are representative of 2independent experiments; n=3. In FIG. 1E-1H, C57BL6/J mice were infectedwith Armstrong strain LCMV, and splenocytes were analyzed 7 days afterinfection. Data are representative of 2 independent experiments; n=5.FIG. 1E-1F show flow cytometry histogram representative of TIGITexpression by naïve (CD44^(low) CD62L^(high)) and effector memory(CD44^(high) CD62L^(low)) CD4+ and CD8+ T cells. Quantitation of TIGITMFI is also shown. ***, P<0.001. FIG. 1G-1H show flow cytometryhistogram representative of TIGIT expression by PD-1^(high) andPD-1^(low) effector memory CD8+ T cells. Quantitation of TIGIT MFI isalso shown. ***, P<0.001. FIG. 1I-1J show that C57BL6/J mice werebriefly depleted of CD4+ T cells and infected with Clone 13 strain LCMV.Splenocytes were analyzed 42 days after infection. Flow cytometryhistogram representative of TIGIT expression by naïve (CD44^(low)CD62L^(high)) central memory (CD44^(high) CD62L^(high)), and effectormemory (CD44^(high) CD62L^(low)) CD8+ T cells. Quantitation of TIGIT MFIis also shown. ***, P<0.001. Data are representative of 2 independentexperiments; n=5. Error bars depict the standard error of the mean.

FIG. 2 shows the design of TIGIT^(loxP/loxP) mice. Exon 1 of TIGIT wasflanked by loxP sites using standard techniques.

FIG. 3A-3L show that TIGIT-deficient CD8⁺ and CD4⁺ T cells respondnormally to acute viral infection. TIGIT^(fl/fl) CD4^(cre) (CKO) andTIGIT^(fl/fl) littermates (WT) were infected with Armstrong strain LCMV.Splenocytes were analyzed 7 days after infection. Data arerepresentative of two independent experiments; n=5. FIG. 3A-3C showrepresentative FACS plots gated on CD8⁺ T cells, with activated(CD44^(high)) cells boxed. Quantitation of activated CD8⁺ T cells as apercentage of total CD8⁺ T cells. FIG. 3D-3F show representative FACSplots gated on CD8⁺ T cells after stimulation in vitro, withIFNγ-producing cells boxed. Quantitation of IFNg-producing cells as apercentage of total CD8⁺ T cells. FIG. 3G-3I show representative FACSplots gated on CD4⁺ T cells, with activated (CD44^(high)) cells boxed.Quantitation of activated CD4⁺ T cells as a percentage of total CD4⁺ Tcells. FIG. 3J-3L show representative FACS plots gated on CD4⁺ T cellsafter stimulation in vitro, with IFNg-producing cells boxed.Quantitation of IFNg-producing cells as a percentage of total CD4⁺ Tcells. Error bars depict the standard error of the mean.

FIG. 4A-4X show that TIGIT and PD-1 synergistically regulate theeffector function of exhausted T cells in vivo. In FIG. 4A-4M,TIGIT^(fl/fl) CD4-cre-(WT) and TIGIT^(fl/fl) CD4-cre+(CKO) mice werebriefly depleted of CD4+ T cells and infected with Clone 13 strain LCMV.Splenocytes and liver viral titers were analyzed 42 days afterinfection. Data are representative of 2 independent experiments, andn=6-9 per group. FIG. 4A-4C depict representative FACS plots gated onCD8+ T cells, with activated cells (CD44^(high) CD62L^(low)) boxed.Quantitation of activated cells as a percentage of total CD8+ T cells.FIG. 4B-4F depict representative FACS plots gated on CD8+ T cells afterstimulation in vitro, with IFNγ+ cells boxed. Quantitation ofIFNγ-producing cells as a percentage of CD8+ T cells. FIG. 4G-4I depictrepresentative FACS plots gated on CD4+ T cells, with activated cells(CD44^(high) CD62L^(low)) boxed. Quantitation of activated cells as apercentage of total CD4+ T cells. FIG. 4J-4L depict representative FACSplots gated on CD4+ T cells after stimulation in vitro, with IFNγ+ cellsboxed. Quantitation of IFNγ-producing cells as a percentage of CD4+ Tcells. FIG. 4M depicts quantitation of liver LCMV titers. ***, P<0.0001.In FIG. 4N-4X, C57BL6/J mice were briefly depleted of CD4+T cells andinfected with Clone 13 strain LCMV. Mice were treated withisotype-matched control, anti-PD-L1, anti-TIGIT, or anti-PD-L1+anti-TIGIT antibodies starting 28 days after infection. Splenocytes andliver viral titers were analyzed 42 days after infection. Data arerepresentative of 2 independent experiments; n=10. FIG. 4N-4R depictrepresentative FACS plots gated on CD8+ T cells, with activated cells(CD44^(high) CD62L^(low)) boxed. Quantitation of activated cells as apercentage of total CD8+ T cells. ***, P<0.0001. FIG. 4S-4W depictrepresentative FACS plots gated on activated CD8+ T cells afterstimulation in vitro, with IFNγ+ cells boxed. Quantitation ofIFNγ-producing cells as a percentage of activated CD8+ T cells. *.P=0.0352. **, P=0.0047. FIG. 4X depicts quantitation of liver LCMVtiters. *, P=0.0106. **, P=0.0047. Error bars depict the standard errorof the mean.

FIG. 5A-5J show TIGIT/PD-L1 co-blockade enhances CD4+ T cell effectorfunction during chronic viral infection. C57BL6/J mice were depleted ofCD4+ T cells and infected with Clone 13 strain LCMV. Mice were treatedwith isotype control, anti-PD-L1, anti-TIGIT, or anti-PD-L1+ anti-TIGITantibodies from 28 days after infection. Splenocytes and liver viraltiters were analyzed 42 days after infection. Data are representative of2 independent experiments; n=10. FIG. 5A-5E depict representative FACSplots gated on CD4+ T cells, with activated cells (CD44^(high)CD62L^(low)) boxed. Quantitation of activated CD4+ T cells as apercentage of total CD4+ T cells. FIG. 5F-5J depict representative FACSplots gated on CD4+ T cells after stimulation in vitro, withIFNγ-producing cells boxed. Quantitation of IFNγ-producing cells as apercentage of total CD4+ T cells. *, P=0.019. Error bars depict thestandard error of the mean.

FIG. 6A-6H show TIGIT expression is elevated in human breast cancer andcorrelated with expression of CD8 and inhibitory co-receptors. Breastcancer gene expression microarray data generated by the Cancer GeneAtlas Network was analyzed. Gene expression data is normalized andexpressed as relative ratios (log 2). FIG. 6A-6B depict TIGIT expressionin normal and all breast tumor samples (left) and in breast tumorsubtypes (right). ***, P=6×10-12. Box and whisker plots are shown. FIG.6C depicts correlation of TIGIT and CD3ε expression. R2=0.61. FIG. 6D-6Edepict the correlation of TIGIT and CD8α (left, R2=0.80) or CD4 (right,R2=0.42). FIG. 6F-6H depict the correlation of TIGIT and PD-1 (left,R²=0.87), LAG3 (center, R²=0.80), and CTLA4 (right, R²=0.76).

FIG. 7A-7P show that TIGIT and PD-1 inhibit anti-tumor T cell responses.In FIG. 7A-7D, BALB/C mice were inoculated with CT26 colorectalcarcinoma cells. Splenocytes and tumor-infiltrating lymphocytes (TILs)were analyzed 14 days after inoculation, when tumors had reachedapproximately 200 mm³ in size. Data are representative of oneexperiment; n=6. FIG. 7A-7B depict flow cytometry histogramrepresentative of TIGIT expression by splenic and tumor-infiltratingCD8+ T cells. Quantitation of TIGIT MFI is also shown. **, P=0.0023.FIG. 7C-7D depict flow cytometry histogram representative of TIGITexpression by splenic and tumor-infiltrating CD4+ T cells. Quantitationof TIGIT MFI is also shown. ***, P=0.0002. In FIG. 7E-7K, BALB/C micewere inoculated with CT26 colorectal carcinoma cells. When tumorsreached approximately 200 mm3 in size, mice were treated with isotypecontrol, anti-PD-L1, anti-TIGIT, or anti-PD-L1+ anti-TIGIT antibodiesfor three weeks. Data are representative of two independent experiments;n=10-20 (FIG. 7E-7F) or 7-10 (FIG. 7G-7K). FIG. 7E depicts median CT26tumor volumes over time. FIG. 7F depicts mouse survival. FIG. 7G-7K showthat approximately 60 days after initial inoculation, mice in completeremission (CR) that had received anti-TIGIT+ anti-PD-L1, as well asnaïve BALB/c mice, were inoculated with CT26 cells in their leftthoracic flanks and inoculated with EMT6 breast carcinoma cells in theirmammary fat pads. Median (left) and individual (right) tumor volumes forCT26 (squares) and EMT6 (triangles) in CR mice (purple and green) andnaïve mice (black and orange) tumors are shown. FIG. 7L-7P show thatmice were inoculated with CT26 tumors and treated as in FIG. 7E.Tumor-infiltrating and tumor-draining lymph node resident T cells wereanalyzed by flow cytometry. Representative FACS plots of CD8+ TILs afterstimulation in vitro, with IFNγ-producing cells boxed. Quantitation ofIFNγ-producing CD8+ TILs as a percentage of total CD8+ TILs. ***,P=0.0003. Data are representative of two independent experiments; n=5.Error bars depict the standard error of the mean.

FIG. 8A-8D show CT26 tumor-infiltrating lymphocyte TIGIT expression iscorrelated with Tim-3 expression. BALB/C mice were inoculated with CT26colorectal carcinoma cells. Splenocytes and tumor-infiltratinglymphocytes (TILs) were analyzed approximately 14 days afterinoculation, when tumors had reached approximately 200 mm³ in size. Dataare representative of one experiment; n=6. FIG. 8A-8B depictrepresentative histogram of TIGIT expression by splenic andtumor-infiltrating CD8⁺ T cells. Quantitation of TIGIT MFI. **,P=0.0026. FIG. 8C-8D depict representative histogram of TIGIT expressionby splenic and tumor-infiltrating CD4⁺ T cells. Quantitation of TIGITMFI. ***, P<0.0001. Error bars depict the standard error of the mean.

FIG. 9A-9D show MC38 tumor-infiltrating lymphocyte TIGIT expression iscorrelated with PD-1 and Tim-3 expression. C57BL6/J mice were inoculatedwith MC38 colorectal carcinoma cells. Splenocytes and tumor-infiltratinglymphocytes (TILs) were analyzed approximately 14 days afterinoculation, when tumors had reached approximately 200 mm³ in size. Dataare representative of one experiment; n=5. FIG. 9A-9B depictrepresentative histogram of TIGIT expression by splenic andtumor-infiltrating CD8⁺ T cells. Quantitation of TIGIT MFI. ***,P<0.0001. FIG. 9C-9D depict representative histogram of TIGIT expressionby splenic and tumor-infiltrating CD4⁺ T cells. Quantitation of TIGITMFI. *, P=0.0136. **, P=0.0029. Error bars depict the standard error ofthe mean.

FIG. 10 shows CT26 tumor growth in mice treated with anti-PD-L1 and/oranti-TIGIT. Naïve BALB/c mice were inoculated with CT26 tumor cells andtreated with anti-PD-L1 and/or anti-TIGIT or isotype-matched controlantibodies, as described in FIG. 4D-4F. Tumor volumes over time forindividual mice in each treatment group are shown. Data arerepresentative of two independent experiments.

FIG. 11A-11F show flow cytometric analysis of CD4⁺ TILs andtumor-draining lymph node T cells. BALB/C mice were inoculated with CT26colorectal carcinoma cells. When tumors reached approximately 200 mm³ insize, mice were treated with isotype control, anti-PD-L1, anti-TIGIT, oranti-PD-L1+ anti-TIGIT antibodies for 7 days. Tumors and tumor-draininglymph nodes were harvested. Data are representative of two independentexperiments; n=5. Representative FACS plots gated on tumor-draininglymph node CD8⁺ T cells after stimulation in vitro, with IFNγ-producingcells boxed. Quantitation of IFNγ⁺ cells as a percentage of total CD8⁺ Tcells. ***, P<0.001. Quantitation of CD8⁺ T cells as a percentage oftotal TILs. **, P=0.0065. Quantitation of activated (CD44^(high)CD62L^(low)) CD8⁺ T cells as a percentage of total CD8⁺ TILs. *,P=0.012. Quantitation of CD8⁺ T cells as a percentage of totaltumor-draining lymph node cells. Quantitation of activated CD8⁺ T cellsas a percentage of total CD8⁺ T cells in the tumor-draining lymph node.*, P<0.05. FIG. 11C depicts quantitation of CD4⁺ T cells as a percentageof total TILs. *, P=0.016. FIG. 11D depicts quantitation of activatedCD4⁺ T cells as a percentage of total CD4⁺ TILs. FIG. 11E depictsquantitation of CD4⁺ T cells as a percentage of total tumor-draininglymph node cells. FIG. 11F depicts quantitation of activated CD4⁺ Tcells as a percentage of total CD4⁺ T cells in the tumor-draining lymphnode. FIG. 11A depicts quantitation of IFNγ⁺ cells as a percentage ofCD4⁺ TILs after stimulation in vitro. FIG. 11B depicts quantitation ofIFNγ⁺ cells as a percentage of CD4⁺ T cells in the tumor-draining lymphnode after stimulation in vitro. Error bars depict the standard error ofthe mean.

FIG. 12A-12C show further flow cytometric analysis of CD8⁺ TILs. BALB/Cmice were inoculated with CT26 colorectal carcinoma cells and treatedwith isotype control, anti-PD-L1, anti-TIGIT, or anti-PD-L1+ anti-TIGITantibodies as described in FIG. 4A-4X. Tumors were harvested after 7days of treatment and analyzed by flow cytometry. Data arerepresentative of two independent experiments; n=5. FIG. 12A depictsquantitation of TNFα⁺ cells as a percentage of total CD8⁺ TILs. **,P<0.01. FIG. 12B depicts quantitation of CD8⁺ TILs as a percentage oftotal TILs. **, P<0.01. FIG. 12C depicts quantitation of activated(CD44^(high) CD62L^(low)) CD8⁺ TILs as a percentage of total CD8⁺ TILs.*, P<0.05. Error bars depict the standard error of the mean.

FIG. 13A-13I show flow cytometric analysis of tumor-draining lymph noderesident CD8⁺ T cells. BALB/C mice were inoculated with CT26 colorectalcarcinoma cells and treated with isotype control, anti-PD-L1,anti-TIGIT, or anti-PD-L1+ anti-TIGIT antibodies as described in FIG.4A-4X. Tumor-draining lymph nodes were harvested after 7 days oftreatment and analyzed by flow cytometry. Data are representative of twoindependent experiments; n=5. FIG. 13A-13E depict representative FACSplots gated on tumor-draining lymph node resident CD8⁺ T cells afterstimulation in vitro, with IFNγ-producing cells boxed. Quantitation ofIFNγ⁺ cells as a percentage of total CD8⁺ T cells. ***, P<0.001. FIG.13F depicts quantitation of CD8⁺ T cells as a percentage of total cellsin the tumor-draining lymph node. FIG. 13G depicts quantitation ofactivated (CD44^(high) CD62L^(low)) CD8⁺ T cells as a percentage oftotal CD8⁺ T cells. *, P<0.05. Error bars depict the standard error ofthe mean. FIG. 13H-13I depict quantitation of TNFα-producing cells as apercentage of total tumor-draining lymph node CD8⁺ T cells.

FIG. 14 shows co-expression of CD226 and TIGIT by tumor-infiltratingCD8+ T cells. C57BL6/J mice were inoculated with MC38 colorectalcarcinoma cells. Splenocytes and tumor-infiltrating lymphocytes (TILs)were analyzed approximately 14 days after inoculation, when tumors hadreached approximately 200 mm³ in size. Representative histogram of CD226expression by splenic B cells (gray), splenic CD8+ T cells (blue), andTIGIT+ tumor-infiltrating CD8+ T cells (red). Data are representative oftwo independent experiments; n=5.

FIG. 15 shows CD226 and TIGIT Co-Immunoprecipate (co-IP) on transfectedcells. COS7 cells were co-transfected with expression plasmidscontaining the cDNA for either TIGIT-HA (5 ng) or CD226-Flag (10 ng)tagged proteins, or a control plasmid (pRK). Following transfection, thecells were washed and centrifuged and cell pellets lysed. The resultantsupernatant was pre-cleared and centrifuged and then equally split intotwo tubes and immuno-precipitated with either an anti-HA or an anti-flagusing standard procedures. The immune-precipitated proteins weresubjected to SDS-PAGE and western blotted. Western blots were probedwith either anti-Flag-HRP or anti-HA-HRP.

FIG. 16 shows TIGIT and CD226 interact in primary CD8+ T cells.MACS-enriched splenic C57BL6/J CD8+ T cells were stimulated withplate-bound anti-CD3 and anti-CD28 antibodies and recombinant IL-2 for48 hours and lysed. Cell lysates were immunoprecipitated with anti-TIGITand probed with anti-CD226. Lanes: molecular weight ladder (1), input(2), co-immunoprecipitation flow-through (3), and co-immunoprecipitate.Arrow denotes the expected molecular weight of CD226.

FIG. 17A-17F show that detection of TIGIT/CD226 interaction by TR-FRET.FIG. 17A-17B depict the dissociation of Flag-ST-CD226 homodimers byHA-TIGIT. FRET ratio between Flag-ST-CD226 measured on COS-7 cellsexpressing a constant amount of Flag-ST-CD226 and increasingconcentrations of HA-TIGIT. FIG. 17C depicts FRET ratio betweenFlag-ST-CD226 recorded after a 15-min incubation of either PBS (whitebar) or anti-TIGIT antibody (black bar). FIG. 17D-17E depict theassociation of Flag-ST-CD226 with HA-TIGIT. FRET intensity betweenFlag-ST-CD226 and HA-TIGIT over the Flag-ST-CD226 expression as measuredby an anti-Flag ELISA on the same batch of transfected COS-7 cells. FIG.17F depicts FRET variation between Flag-ST-CD226 and HA-TIGIT after a15-min incubation of PBS (white bar) or anti-TIGIT antibody (black bar).Data in FIG. 17A and FIG. 17D are representative of 4 independentexperiments, each performed in triplicate. Data in FIG. 17C and FIG. 17Fare representative of 2 independent experiments, each performed intriplicate.

FIG. 18 shows cell surface expression of Flag-ST-CD226 and HA-TIGIT.Anti-Flag and anti-HA ELISA on intact COS-7 cells expressing theindicated tagged-constructs. Data are representative of 3 independentexperiments, each performed in triplicate.

FIG. 19A-19D show that CD226 blockade reverses the enhanced anti-viral Tcell response induced by TIGIT/PD-L1 co-blockade. In FIG. 19A-19D,C57BL6/J mice were briefly depleted of CD4⁺ T cells and infected withClone 13 strain LCMV. Mice were treated with isotype-matched control,anti-CD226, anti-PD-L1+ anti-TIGIT, or anti-PD-L1+ anti-TIGIT+anti-CD226 antibodies starting 28 days after infection. Splenocytes andliver viral titers were analyzed 42 days after infection. FIG. 19Adepicts quantitation of CD8⁺ T cells as a percentage of splenocytes.FIG. 19B depicts quantitation of activated CD8⁺ T cells as a percentageof total CD8⁺ T cells. ***, P<0.001. FIG. 19C depicts quantitation ofIFNg-producing cells as a percentage of activated CD8⁺ T cells. ***,P<0.001. FIG. 19D depicts quantitation of liver LCMV titers. ***,P<0.001. Error bars depict the standard error of the mean.

FIG. 20A-20N show that TIGIT expression is elevated in human cancer andstrongly correlated with CD8 and PD-1. Gene expression analyses of humancancers were performed as described in Example 11. Scatter plots showper-gene count data, normalized by library size. Box and whisker plotsshow the variance stabilized expression ratio of TIGIT and CD3e. FIG.20A-20B depict the correlation of TIGIT and CD3e RNA expression in LUSC(grey) and normal lung (black). ρ=0.86. Quantification of TIGIT/CD3eexpression ratios is also shown. LUSC ratio increase=372%. ***,P=1.46×10⁴⁶. FIG. 20C-20D depict the correlation of TIGIT and CD3e RNAexpression in COAD (grey) and normal colon (black). ρ=0.83.Quantification of TIGIT/CD3e expression ratios is also shown. COAD ratioincrease=116%. ***, P=3.66×10⁻⁶. FIG. 20E-20F depict the correlation ofTIGIT and CD3e RNA expression in UCEC (grey) and normal uterineendrometrium (black). ρ=0.87. Quantification of TIGIT/CD3e expressionratios is also shown. UCEC ratio increase=419%. ***, P=7.41×10⁻⁵. FIG.20G-20H depict the correlation of TIGIT and CD3e RNA expression in BRCA(grey) and normal breast (black). ρ=0.82. Quantification of TIGIT/CD3eexpression ratios is also shown. BRCA ratio increase=313%. ***,P=4.6×10⁻⁴⁴. FIG. 20I-20J depict the correlation of TIGIT and CD3e RNAexpression in kidney renal clear cell carcinoma (grey) and normal kidney(black). ρ=0.94. Quantification of TIGIT/CD3e expression ratios is alsoshown. FIG. 20K-20L depict the correlation of TIGIT and CD8A (left) orTIGIT and CD4 (right) in lung squamous cell carcinoma (grey) and normallung (black). ρ=0.77 and 0.48 respectively. FIG. 20M depicts thecorrelation of TIGIT and PD-1 (Pdcd1) in lung squamous cell carcinoma(grey) and normal lung (black). ρ=0.82. FIG. 20N depicts the correlationof TIGIT and CD226 in lung squamous cell carcinoma (red) and normal lung(black). ρ=0.64.

FIG. 21 shows analysis of T cell-associated gene expression in LungSquamous Cell Carcinoma (LUSC). Gene expression in LUSC and normaltissue samples was analyzed as described in Example 11 and a heat map ofthe genes best correlated with the gene signature in LUSC samples wasgenerated. Genes and samples were both clustered using hierarchicalclustering using Ward linkage on the Euclidean distance matrix for thecentered and scaled expression data.

FIG. 22A-22P shows that TIGIT and PD-1 are coordinately expressed byhuman and murine tumor-infiltrating lymphocytes. FIG. 22A-22H showsanalysis of lymphocytes from a freshly resected human NSCLC tumor,tumor-matched peripheral blood, and normal donor peripheral blood. Dataare representative of two independently analyzed tumors. FIG. 22A-22Cdepicts representative FACS plots representative of TIGIT expression byperipheral and tumor-infiltrating CD8⁺ T cells, with TIGIT⁺ cells boxed.FIG. 22D-22F depict representative FACS plots representative of TIGITexpression by peripheral and tumor-infiltrating CD4⁺ T cells, withTIGIT⁺ cells boxed. FIG. 22G-22H depict flow cytometry histogramrepresentative of TIGIT expression by PD-1^(high) (red) and PD-1^(low)(blue) NSCLC-infiltrating CD8⁺ (left) and CD4⁺ (right) T cells. In FIG.22I-22P, BALB/C mice were inoculated with syngeneic CT26 colorectalcarcinoma cells. Splenocytes and tumor-infiltrating lymphocytes (TILs)were analyzed 14 days after inoculation, when tumors had reachedapproximately 200 mm³ in size. Data are representative of twoindependent experiments; n=5-6. FIG. 22I-22J depict representative FACSplot of TIGIT expression by tumor-infiltrating CD8⁺ T cells, with TIGIT⁺cells boxed. FIG. 22K-22L depict representative FACS plot of TIGITexpression by tumor-infiltrating CD4⁺ T cells, with TIGIT⁺ cells boxed.Quantitation of the frequency of TIGIT⁺ T cells as a percentage of all Tcells. *, P=0.0134. ***, P<0.0001. FIG. 22M-22N depict flow cytometryhistogram representative of TIGIT expression by PD-1^(high) andPD-1^(low) tumor-infiltrating CD8⁺ T cells and by splenic CD8⁺ T cells.Quantitation of TIGIT MFI is also shown. **, P=0.0023. FIG. 220-22Pdepict flow cytometry histogram representative of TIGIT expression byPD-1^(high) and PD-1^(low) tumor-infiltrating CD4⁺ T cells and bysplenic CD4⁺ T cells. Quantitation of TIGIT MFI is also shown. ***,P=0.0002. Error bars depict the standard error of the mean.

FIG. 23A-23H show characterization of TIGIT expression by humantumor-infiltrating T cells. FIG. 23A-23D depict FACS plots showing TIGITexpression by NSCLC tumor-infiltrating CD8+ and CD4+ T cells (FIG.23A-23B) and by donor-matched PBMC CD8+ and CD4+ T cells (FIG. 23C-23D),with TIGIT+ cells boxed. FIG. 23E-23H depict FACS plots showing TIGITexpression by CRC tumor-infiltrating CD8+ and CD4+ T cells (FIG.23E-23F) and by donor-matched PBMC CD8+ and CD4+ T cells (FIG. 23G-23H),with TIGIT+ cells boxed.

FIG. 24 shows that the TIGIT:CD226 interaction is not driven by PVRTIGIT:CD226 and TIGIT Q56R:CD226 interactions were detected by TR-FRETand the FRET ratio between Flag-ST-CD226 and HA-TIGIT or HA-TIGIT Q56Rshows that WT and Q56R TIGIT bind CD226 with the same efficacy. Data arerepresentative of three independent experiments performed in triplicate.

FIG. 25A-25G show that efficacy of TIGIT/PD-L1 antibody co-blockade inmice bearing MC38 tumors. In FIG. 25A-25G, MC38 tumor-bearing mice weregenerated as above and treated with blocking antibodies against PD-L1(red), TIGIT (blue), TIGIT and PD-L1 (purple) or isotype-matched controlantibodies (black) for three weeks. N=10 (control, anti-PD-L1 alone,anti-TIGIT alone) or 20 (anti-TIGIT+ anti-PD-L1). FIG. 25A-25E depictmedian (left) and individual (right) MC38 tumor volumes over time. FIG.25F depicts MC38 tumor volumes after 14 days of antibody treatment. ***,P=0.0005. **, P=0.0093. *, P=0.0433. FIG. 25G depicts mouse survivalover time. Error bars depict the standard error of the mean.

FIG. 26A-26L show that further characterization of TIGIT expression bymurine tumor-infiltrating T cells. FIG. 26A-26D depict that splenicC57BL6/J CD8⁺ T cells were enriched by MACS and cultured withplate-coated anti-CD3 and anti-CD28 agonist antibodies. Representativehistograms of TIGIT (red) and isotype-matched control (solid gray)staining over time. Quantitation of TIGIT MFI. ***, P<0.001. Stimulatedcells inducibly expressed PD-1 and constitutively expressed CD226 (datanot shown). Data are representative of two independent experiments; n=5.In FIG. 26E-26L, wildtype C57BL6/J mice were subcutaneously inoculatedwith syngeneic MC38 colorectal carcinoma cells. Tumors were allowed togrow without intervention until they reached 150-200 mm³ in size. Dataare representative of two independent experiments; n=5. FIG. 26E-26Fdepict representative FACS plot of tumor-infiltrating CD8⁺ T cells, withTIGIT cells boxed. Quantitation of the frequency of TIGIT cells as apercentage of all tumor-infiltrating or splenic CD8⁺ T cells. ***,P<0.0001. FIG. 26G-26H depict representative FACS plot oftumor-infiltrating CD4⁺ T cells, with TIGIT cells boxed. Quantitation ofthe frequency of TIGIT cells as a percentage of all tumor-infiltratingor splenic CD4⁺ T cells. ***, P<0.0001. FIG. 26I-26J depictrepresentative histogram of TIGIT expression by PD-1^(high) andPD-1^(low) tumor-infiltrating CD8⁺ T cells (red and blue, respectively)and by splenic CD8⁺ T cells (gray). Quantitation of TIGIT MFI. ***,P<0.0001. FIG. 26K-26L depict representative histogram of TIGITexpression by PD-1^(high) and PD-1^(low) tumor-infiltrating CD4⁺ T cellsand by splenic CD4⁺ T cells. Quantitation of TIGIT MFI. *, P=0.0136. **,P=0.0029. Error bars depict the standard error of the mean.

FIG. 27A-27C show that tumor-infiltrating CD8⁺ and CD4⁺ T cells maintaina high level of CD226 expression. Wildtype BALB/c mice were inoculatedwith CT26 tumor cells as described herein. After tumors have grown toapproximately 150-200 mm³ in size, tumors and spleens were analyzed byflow cytometry. FIG. 27A depicts quantitation of CD226⁺ CD8⁺ T cells,CD4⁺ T cells, and non-T cells, as a percentage of all CD8⁺ T cells, CD4⁺T cells, and non-T cells respectively. FIG. 27B-27C depictrepresentative histograms of CD226 expression in tumor and spleen. Dataare representative of two independent experiments; n=5. Error barsdepict the standard error of the mean.

FIG. 28A-28K show that TIGIT suppression of CD8⁺ T cell responses isdependent on CD226. BALB/C mice were subcutaneously inoculated with CT26colorectal carcinoma cells in their right thoracic flanks. When tumorsreached approximately 200 mm³ in size, mice were treated with isotypecontrol (black), anti-CD226 (orange), anti-PD-L1 (red), anti-TIGIT+anti-PD-L1 (purple), or anti-TIGIT+ anti-PD-L1+ anti-CD226 (green)antibodies for three weeks. Data are representative of one experiment;n=10 (FIG. 28A-28G) or 5 (FIG. 28H-28K). FIG. 28A-28F depict median(left) and individual (right) CT26 tumor volumes over time. FIG. 28Gdepicts mouse survival over time. In FIG. 28H-28K, after 7 days oftreatment, tumor-infiltrating lymphocytes and tumor-draining lymphnode-resident lymphocytes were assessed by flow cytometry. FIG. 28Hdepicts quantitation of IFNγ-producing CD8⁺ TILs as a percentage oftotal CD8⁺ TILs after stimulation in vitro. ** , P<0.01. FIG. 28Idepicts quantitation of IFNγ-producing cells as a percentage of totalCD8⁺ T cells after stimulation in vitro. *, P<0.05. FIG. 28J depictsquantitation of CD8⁺ TILs as a percentage of total TILs. **, P<0.01.FIG. 28K depicts quantitation of CD8⁺ T cells as a percentage of alltumor-draining lymph node-resident lymphocytes. Error bars depict thestandard error of the mean.

FIG. 29A-29H show that TIGIT impairs CD226 function by directlydisrupting CD226 homodimerization. FIG. 29A depicts that CD8⁺ T cellswere MACS-enriched from TIGIT^(fl/fl) CD4^(cre) (CKO) and TIGIT^(fl/fl)CD4^(wt) (WT) littermates and stimulated in the presence of anti-CD226or isotype-matched control antibodies as indicated. H³-thymidine uptakeis shown as a ratio of cells cultured with anti-CD3+PVR-Fc to cellscultured with anti-CD3 alone. **, P=0.0061. ***, P<0.0001. Data arerepresentative of two independent experiments; n=5. FIG. 29B depictsthat wildtype C57BL6/J CD8⁺ T cells were MACS-enriched and stimulated inthe presence of anti-TIGIT, anti-CD226, and/or isotype-matched controlantibodies as indicated. H³-thymidine uptake is shown as a ratio ofcells cultured with anti-CD3+PVR-Fc to cells cultured with anti-CD3alone. ***, P<0.001 in paired t tests. FIG. 29C depicts that primaryhuman CD8⁺ T cells were MACS-enriched from blood and stimulated withsub-optimal levels of plate-bound anti-CD3 in the presence or absence ofhuman recombinant PVR-Fc. Anti-TIGIT antibodies or isotype-matchedcontrol antibodies were added as indicated. Quantitation of ³H-thymidineuptake. **, P=0.0071 and 0.0014 respectively. FIG. 29D depicts that CHOcells were transiently transfected with increasing concentrations ofacceptor and donor FLAG-ST-CD226, as indicated. Quantification of FRETintensity relative to donor emission. Data are representative of threeindependent experiments; n=3. In FIG. 29E-29F, CHO cells weretransiently transfected with FLAG-ST-CD226 and with increasingconcentrations of HA-TIGIT, as indicated. Data are representative of twoor more independent experiments; n=4. Data are normalized to the maximalsignal. FIG. 29E depicts quantification of the CD226:CD226 FRET ratio(FRET ratio 1). FIG. 29F depicts quantification of the TIGIT:CD226 FRETratio (FRET ratio 2). FIG. 29G depicts anti-FLAG (left) and anti-HA(right) immunoblots performed on either anti-FLAG or anti-HAimmunoprecipitates prepared from COS-7 cells transfected with either anempty pRK vector or a combination of Flag-CD226 and HA-TIGIT. Data arerepresentative of two independent experiments. FIG. 29H depictsquantification of the TIGIT:CD226 FRET ratio after incubation with PBS(white) or anti-TIGIT antibodies (red). ***, P<0.001. Data arerepresentative of 4 independent experiments; n=3. Error bars depict thestandard error of the mean.

FIG. 30 shows that primary human T cells were MACS-enriched from bloodand stimulated with anti-CD3 and anti-CD28. TIGIT⁺ and TIGIT⁻ cells weresorted, rested, re-stimulated, and labeled for FRET with the antibodiesindicated. Data are representative of two independent experiments. ***,P<0.001. Error bars depict the standard error of the mean.

FIG. 31A-31G show that TIGIT and PD-1 co-blockade does not restore theeffector function of exhausted CD4⁺ T cells during chronic viralinfection. FIG. 31A depicts quantitation of CD8⁺ T cells as a percentageof all splenocytes. FIG. 31B depicts quantitation of GP33 Pentamer⁺cells as a percentage of all splenic CD8⁺ T cells. **, P=0.0040. FIG.31C-31G depict representative FACS plots gated on gp33 pentamer⁺ CD8⁺ Tcells after stimulation in vitro, with IFNγ⁺ cells boxed. Quantitationof IFNγ-producing cells as a percentage of all gp33 pentamer⁺ CD8⁺ Tcells. *, P=0.0319. **, P=0.0030. Error bars depict the standard errorof the mean.

FIG. 32A-32I show that TIGIT/PD-L1 co-blockade efficacy is dependent onCD8⁺ T cells. In FIG. 32A-32E, wildtype BALB/c mice were inoculated withCT26 tumors as described in FIG. 7A-7P. When tumors reached 100-150 mm³in size, mice were temporarily depleted of CD8⁺ T cells and treated withanti-TIGIT+ anti-PD-L1. Data are representative of one experiment;n=10/group. FIG. 32A-32D depict median (left) and individual (right)CT26 tumor volumes over time. FIG. 32E depicts quantitation of CT26tumor volumes 17 days after the start of treatment. ***, P=0.0004. InFIG. 32F-32I, wildtype BALB/c mice were inoculated with CT26 tumors andtreated with anti-TIGIT+ anti-PD-L1 and subsequently re-challenged withCT26 tumors with temporary depletion of CD8⁺ T cells at the time ofre-challenge. Data are representative of two independent experiments;n=5. FIG. 32F-32I depict median (left) and individual (right) CT26 tumorvolumes over time. Error bars depict the standard error of the mean.

FIG. 33 shows that PVR expression on tumor cells is dispensable forTIGIT/PD-L1 co-blockade efficacy. Wildtype BALB/c mice were inoculatedwith wildtype or PVR-deficient (PVR.KO) tumors as described. When tumorsreached 150-200 mm³ in size, mice were treated with anti-TIGIT+anti-PD-L1 or isotype-matched control antibodies. Data arerepresentative of one experiment; n=10/group. FIG. 33 depicts median(left) and individual (right) CT26 tumor volumes over time.

FIG. 34 shows the efficacy of TIGIT/PD-L1 antibody co-blockade in micebearing EMT6 tumors. EMT6 tumor-bearing mice were generated as above andtreated with blocking antibodies against PD-L1 (red), TIGIT (blue),TIGIT and PD-L1 (purple) or isotype-matched control antibodies (black)for three weeks. N=10 (control, anti-PD-L1 alone, anti-TIGIT alone) or20 (anti-TIGIT+ anti-PD-L1). FIG. 34 depicts median (left) andindividual (right) EMT6 tumor volumes over time.

FIG. 35A-35D show that TIGIT regulates tumor-infiltrating CD8⁺ T celleffector function. BALB/C mice were subcutaneously inoculated with CT26colorectal carcinoma cells in their right thoracic flanks and treatedwith anti-PD-L1, anti-TIGIT, or anti-PD-L1+ anti-TIGIT, as described inFIG. 7A-7P. Tumor-draining lymph node (dLN) resident andtumor-infiltrating T cells were analyzed by flow cytometry 7 days afterthe start of treatment. Data are representative of two independentexperiments; n=5. FIG. 35A-35B depict quantitation of IFNγ/TNFαdual-producing dLN resident CD8⁺ and CD4⁺ T cells as percentages oftotal dLN resident CD8⁺ and CD4⁺ T cells respectively. Dual cytokineproduction by unstimulated T cells is also shown. **, P=0.002, 0.003,and 0.001 respectively. FIG. 35C-35D depict quantitation of IFNγ/TNFαdual-producing tumor-infiltrating CD8⁺ and CD4⁺ T cells as percentagesof total tumor-infiltrating CD8⁺ and CD4⁺ T cells respectively. Dualcytokine production by unstimulated T cells is also shown. ***,P<0.0001. Error bars depict the standard error of the mean.

FIG. 36A-36B show analysis of lymphocytes from resected human NSCLCtumors, tumor-matched peripheral blood, and normal donor peripheralblood. Data are pooled from three independently acquired sets ofsamples. FIG. 36A depicts quantitation of TIGIT cells as a percentage ofall CD8⁺ T cells. *, P<0.05. FIG. 36B depicts quantitation of TIGITcells as a percentage of all CD4⁺ T cells.

FIG. 37A-37E show characterization of TIGIT expression in human tumors.FIG. 37A-37C depict representative flow cytometry histograms of TIGITexpression by NSCLC tumor-resident lymphocytes (red, CD45⁺ FSC^(low)),myeloid cells (blue, CD45⁺ FSC^(high)), and non-hematopoietic cells(green, CD45⁻) relative to subset-matched isotype staining (gray). FIG.37D-37E depict gating strategy for PD-1^(high) and PD-1^(low) NSCLCtumor-infiltrating CD8⁺ and CD4⁺ T cells.

DETAILED DESCRIPTION OF THE INVENTION I. General Techniques

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3d edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Current Protocols inMolecular Biology (F. M. Ausubel, et al. eds., (2003)); the seriesMethods in Enzymology (Academic Press, Inc.): PCR 2: A PracticalApproach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and AnimalCell Culture (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; CellBiology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press;Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Celland Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press;Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B.Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbookof Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); GeneTransfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos,eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds.,1994); Current Protocols in Immunology (J. E. Coligan et al., eds.,1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRLPress, 1988-1989); Monoclonal Antibodies: A Practical Approach (P.Shepherd and C. Dean, eds., Oxford University Press, 2000); UsingAntibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold SpringHarbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D.Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principlesand Practice of Oncology (V. T. DeVita et al., eds., J.B. LippincottCompany, 1993).

II. Definitions

The term “PD-1 axis binding antagonist” is a molecule that inhibits theinteraction of a PD-1 axis binding partner with either one or more ofits binding partner, so as to remove T-cell dysfunction resulting fromsignaling on the PD-1 signaling axis—with a result being to restore orenhance T-cell function (e.g., proliferation, cytokine production,target cell killing). As used herein, a PD-1 axis binding antagonistincludes a PD-1 binding antagonist, a PD-L1 binding antagonist and aPD-L2 binding antagonist.

The term “PD-1 binding antagonists” is a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-1 with one or more of its bindingpartners, such as PD-L1, PD-L2. In some embodiments, the PD-1 bindingantagonist is a molecule that inhibits the binding of PD-1 to itsbinding partners. In a specific aspect, the PD-1 binding antagonistinhibits the binding of PD-1 to PD-L1 and/or PD-L2. For example, PD-1binding antagonists include anti-PD-1 antibodies, antigen bindingfragments thereof, immunoadhesins, fusion proteins, oligopeptides andother molecules that decrease, block, inhibit, abrogate or interferewith signal transduction resulting from the interaction of PD-1 withPD-L1 and/or PD-L2. In one embodiment, a PD-1 binding antagonist reducesthe negative co-stimulatory signal mediated by or through cell surfaceproteins expressed on T lymphocytes mediated signaling through PD-1 soas render a dysfunctional T-cell less dysfunctional (e.g., enhancingeffector responses to antigen recognition). In some embodiments, thePD-1 binding antagonist is an anti-PD-1 antibody. In a specific aspect,a PD-1 binding antagonist is MDX-1106 described herein. In anotherspecific aspect, a PD-1 binding antagonist is Merck 3745 describedherein. In another specific aspect, a PD-1 binding antagonist is CT-011described herein. In another specific aspect, a PD-1 binding antagonistis AMP-224 described herein.

The term “PD-L1 binding antagonists” is a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-L1 with either one or more of itsbinding partners, such as PD-1, B7-1. In some embodiments, a PD-L1binding antagonist is a molecule that inhibits the binding of PD-L1 toits binding partners. In a specific aspect, the PD-L1 binding antagonistinhibits binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, thePD-L1 binding antagonists include anti-PD-L1 antibodies, antigen bindingfragments thereof, immunoadhesins, fusion proteins, oligopeptides andother molecules that decrease, block, inhibit, abrogate or interferewith signal transduction resulting from the interaction of PD-L1 withone or more of its binding partners, such as PD-1, B7-1. In oneembodiment, a PD-L1 binding antagonist reduces the negativeco-stimulatory signal mediated by or through cell surface proteinsexpressed on T lymphocytes mediated signaling through PD-L1 so as torender a dysfunctional T-cell less dysfunctional (e.g., enhancingeffector responses to antigen recognition). In some embodiments, a PD-L1binding antagonist is an anti-PD-L1 antibody. In a specific aspect, ananti-PD-L1 antibody is YW243.55.S70 described herein. In anotherspecific aspect, an anti-PD-L1 antibody is MDX-1105 described herein. Instill another specific aspect, an anti-PD-L1 antibody is MPDL3280Adescribed herein. In another specific aspect, an anti-PD-L1 antibody isMEDI 4736 described herein.

The term “PD-L2 binding antagonists” is a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-L2 with either one or more of itsbinding partners, such as PD-1. In some embodiments, a PD-L2 bindingantagonist is a molecule that inhibits the binding of PD-L2 to itsbinding partners. In a specific aspect, the PD-L2 binding antagonistinhibits binding of PD-L2 to PD-1. In some embodiments, the PD-L2antagonists include anti-PD-L2 antibodies, antigen binding fragmentsthereof, immunoadhesins, fusion proteins, oligopeptides and othermolecules that decrease, block, inhibit, abrogate or interfere withsignal transduction resulting from the interaction of PD-L2 with eitherone or more of its binding partners, such as PD-1. In one embodiment, aPD-L2 binding antagonist reduces the negative co-stimulatory signalmediated by or through cell surface proteins expressed on T lymphocytesmediated signaling through PD-L2 so as render a dysfunctional T-cellless dysfunctional (e.g., enhancing effector responses to antigenrecognition). In some embodiments, a PD-L2 binding antagonist is animmunoadhesin.

The term “aptamer” refers to a nucleic acid molecule that is capable ofbinding to a target molecule, such as a polypeptide. For example, anaptamer of the invention can specifically bind to a TIGIT polypeptide,or to a molecule in a signaling pathway that modulates the expression ofTIGIT. The generation and therapeutic use of aptamers are wellestablished in the art. See, e.g., U.S. Pat. No. 5,475,096, and thetherapeutic efficacy of Macugen® (Eyetech, New York) for treatingage-related macular degeneration.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native polypeptide disclosed herein. In asimilar manner, the term “agonist” is used in the broadest sense andincludes any molecule that mimics a biological activity of a nativepolypeptide disclosed herein. Suitable agonist or antagonist moleculesspecifically include agonist or antagonist antibodies or antibodyfragments, fragments or amino acid sequence variants of nativepolypeptides, peptides, antisense oligonucleotides, small organicmolecules, etc. Methods for identifying agonists or antagonists of apolypeptide may comprise contacting a polypeptide with a candidateagonist or antagonist molecule and measuring a detectable change in oneor more biological activities normally associated with the polypeptide.

The terms “TIGIT antagonist” and “antagonist of TIGIT activity or TIGITexpression” are used interchangeably and refer to a compound thatinterferes with the normal functioning of TIGIT, either by decreasingtranscription or translation of TIGIT-encoding nucleic acid, or byinhibiting or blocking TIGIT polypeptide activity, or both. Examples ofTIGIT antagonists include, but are not limited to, antisensepolynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras,TIGIT-specific aptamers, anti-TIGIT antibodies, TIGIT-binding fragmentsof anti-TIGIT antibodies, TIGIT-binding small molecules, TIGIT-bindingpeptides, and other polypeptides that specifically bind TIGIT(including, but not limited to, TIGIT-binding fragments of one or moreTIGIT ligands, optionally fused to one or more additional domains), suchthat the interaction between the TIGIT antagonist and TIGIT results in areduction or cessation of TIGIT activity or expression. It will beunderstood by one of ordinary skill in the art that in some instances, aTIGIT antagonist may antagonize one TIGIT activity without affectinganother TIGIT activity. For example, a desirable TIGIT antagonist foruse in certain of the methods herein is a TIGIT antagonist thatantagonizes TIGIT activity in response to one of PVR interaction, PVRL3interaction, or PVRL2 interaction, e.g., without affecting or minimallyaffecting any of the other TIGIT interactions.

The terms “PVR antagonist” and “antagonist of PVR activity or PVRexpression” are used interchangeably and refer to a compound thatinterferes with the normal functioning of PVR, either by decreasingtranscription or translation of PVR-encoding nucleic acid, or byinhibiting or blocking PVR polypeptide activity, or both. Examples ofPVR antagonists include, but are not limited to, antisensepolynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras,PVR-specific aptamers, anti-PVR antibodies, PVR-binding fragments ofanti-PVR antibodies, PVR-binding small molecules, PVR-binding peptides,and other polypeptides that specifically bind PVR (including, but notlimited to, PVR-binding fragments of one or more PVR ligands, optionallyfused to one or more additional domains), such that the interactionbetween the PVR antagonist and PVR results in a reduction or cessationof PVR activity or expression. It will be understood by one of ordinaryskill in the art that in some instances, a PVR antagonist may antagonizeone PVR activity without affecting another PVR activity. For example, adesirable PVR antagonist for use in certain of the methods herein is aPVR antagonist that antagonizes PVR activity in response to TIGITinteraction without impacting the PVR-CD96 and/or PVR-CD226interactions.

The term “dysfunction” in the context of immune dysfunction, refers to astate of reduced immune responsiveness to antigenic stimulation. Theterm includes the common elements of both exhaustion and/or anergy inwhich antigen recognition may occur, but the ensuing immune response isineffective to control infection or tumor growth.

The term “dysfunctional”, as used herein, also includes refractory orunresponsive to antigen recognition, specifically, impaired capacity totranslate antigen recognition into down-stream T-cell effectorfunctions, such as proliferation, cytokine production (e.g., IL-2)and/or target cell killing.

The term “anergy” refers to the state of unresponsiveness to antigenstimulation resulting from incomplete or insufficient signals deliveredthrough the T-cell receptor (e.g. increase in intracellular Ca⁺² in theabsence of ras-activation). T cell anergy can also result uponstimulation with antigen in the absence of co-stimulation, resulting inthe cell becoming refractory to subsequent activation by the antigeneven in the context of costimulation. The unresponsive state can oftenbe overridden by the presence of Interleukin-2. Anergic T-cells do notundergo clonal expansion and/or acquire effector functions.

The term “exhaustion” refers to T cell exhaustion as a state of T celldysfunction that arises from sustained TCR signaling that occurs duringmany chronic infections and cancer. It is distinguished from anergy inthat it arises not through incomplete or deficient signaling, but fromsustained signaling. It is defined by poor effector function, sustainedexpression of inhibitory receptors and a transcriptional state distinctfrom that of functional effector or memory T cells. Exhaustion preventsoptimal control of infection and tumors. Exhaustion can result from bothextrinsic negative regulatory pathways (e.g., immunoregulatorycytokines) as well as cell intrinsic negative regulatory (costimulatory)pathways (PD-1, B7-H3, B7-H4, etc.).

“Enhancing T-cell function” means to induce, cause or stimulate a T-cellto have a sustained or amplified biological function, or renew orreactivate exhausted or inactive T-cells. Examples of enhancing T-cellfunction include: increased secretion of γ-interferon from CD8⁺ T-cells,increased proliferation, increased antigen responsiveness (e.g., viral,pathogen, or tumor clearance) relative to such levels before theintervention. In one embodiment, the level of enhancement is as least50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. Themanner of measuring this enhancement is known to one of ordinary skillin the art.

A “T cell dysfunctional disorder” is a disorder or condition of T-cellscharacterized by decreased responsiveness to antigenic stimulation. In aparticular embodiment, a T-cell dysfunctional disorder is a disorderthat is specifically associated with inappropriate increased signalingthrough PD-1. In another embodiment, a T-cell dysfunctional disorder isone in which T-cells are anergic or have decreased ability to secretecytokines, proliferate, or execute cytolytic activity. In a specificaspect, the decreased responsiveness results in ineffective control of apathogen or tumor expressing an immunogen. Examples of T celldysfunctional disorders characterized by T-cell dysfunction includeunresolved acute infection, chronic infection and tumor immunity.

“Tumor immunity” refers to the process in which tumors evade immunerecognition and clearance. Thus, as a therapeutic concept, tumorimmunity is “treated” when such evasion is attenuated, and the tumorsare recognized and attacked by the immune system. Examples of tumorrecognition include tumor binding, tumor shrinkage and tumor clearance.

“Immunogenicity” refers to the ability of a particular substance toprovoke an immune response. Tumors are immunogenic and enhancing tumorimmunogenicity aids in the clearance of the tumor cells by the immuneresponse. Examples of enhancing tumor immunogenicity include but notlimited to treatment with a PD-1 axis binding antagonist (e.g.,anti-PD-L1 antibodies and a TIGIT inhibitor (e.g., anti-TIGITantibodies).

“Sustained response” refers to the sustained effect on reducing tumorgrowth after cessation of a treatment. For example, the tumor size mayremain to be the same or smaller as compared to the size at thebeginning of the administration phase. In some embodiments, thesustained response has a duration at least the same as the treatmentduration, at least 1.5×, 2.0×, 2.5×, or 3.0× length of the treatmentduration.

The term “antibody” includes monoclonal antibodies (including fulllength antibodies which have an immunoglobulin Fc region), antibodycompositions with polyepitopic specificity, multispecific antibodies(e.g., bispecific antibodies, diabodies, and single-chain molecules, aswell as antibody fragments (e.g., Fab, F(ab′)₂, and Fv). The term“immunoglobulin” (Ig) is used interchangeably with “antibody” herein.

The basic 4-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains. An IgM antibody consists of 5 of the basic heterotetramer unitsalong with an additional polypeptide called a J chain, and contains 10antigen binding sites, while IgA antibodies comprise from 2-5 of thebasic 4-chain units which can polymerize to form polyvalent assemblagesin combination with the J chain. In the case of IgGs, the 4-chain unitis generally about 150,000 daltons. Each L chain is linked to an H chainby one covalent disulfide bond, while the two H chains are linked toeach other by one or more disulfide bonds depending on the H chainisotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. Each H chain has at the N-terminus, a variable domain(V_(H)) followed by three constant domains (C_(H)) for each of the α andγ chains and four C_(H) domains for μ and ε isotypes. Each L chain hasat the N-terminus, a variable domain (V_(L)) followed by a constantdomain at its other end. The V_(L) is aligned with the V_(H) and theC_(L) is aligned with the first constant domain of the heavy chain(C_(H)1). Particular amino acid residues are believed to form aninterface between the light chain and heavy chain variable domains. Thepairing of a V_(H) and V_(L) together forms a single antigen-bindingsite. For the structure and properties of the different classes ofantibodies, see e.g., Basic and Clinical Immunology, 8th Edition, DanielP. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange,Norwalk, Conn., 1994, page 71 and Chapter 6. The L chain from anyvertebrate species can be assigned to one of two clearly distinct types,called kappa and lambda, based on the amino acid sequences of theirconstant domains. Depending on the amino acid sequence of the constantdomain of their heavy chains (CH), immunoglobulins can be assigned todifferent classes or isotypes. There are five classes ofimmunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chainsdesignated α, δ, ε, γ and μ, respectively. The γ and α classes arefurther divided into subclasses on the basis of relatively minordifferences in the CH sequence and function, e.g., humans express thefollowing subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domains of the heavy chain and light chain may be referred toas “VH” and “VL”, respectively. These domains are generally the mostvariable parts of the antibody (relative to other antibodies of the sameclass) and contain the antigen binding sites.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. The Vdomain mediates antigen binding and defines the specificity of aparticular antibody for its particular antigen. However, the variabilityis not evenly distributed across the entire span of the variabledomains. Instead, it is concentrated in three segments calledhypervariable regions (HVRs) both in the light-chain and the heavy chainvariable domains. The more highly conserved portions of variable domainsare called the framework regions (FR). The variable domains of nativeheavy and light chains each comprise four FR regions, largely adopting abeta-sheet configuration, connected by three HVRs, which form loopsconnecting, and in some cases forming part of, the beta-sheet structure.The HVRs in each chain are held together in close proximity by the FRregions and, with the HVRs from the other chain, contribute to theformation of the antigen binding site of antibodies (see Kabat et al.,Sequences of Immunological Interest, Fifth Edition, National Instituteof Health, Bethesda, Md. (1991)). The constant domains are not involveddirectly in the binding of antibody to an antigen, but exhibit variouseffector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations and/orpost-translation modifications (e.g., isomerizations, amidations) thatmay be present in minor amounts. Monoclonal antibodies are highlyspecific, being directed against a single antigenic site. In contrast topolyclonal antibody preparations which typically include differentantibodies directed against different determinants (epitopes), eachmonoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by a variety of techniques,including, for example, the hybridoma method (e.g., Kohler andMilstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (ColdSpring Harbor Laboratory Press, 2^(nd) ed. 1988); Hammerling et al., in:Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.,1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567),phage-display technologies (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhuet al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol.340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004), and technologies for producing human or human-likeantibodies in animals that have parts or all of the human immunoglobulinloci or genes encoding human immunoglobulin sequences (see, e.g., WO1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits etal., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al.,Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33(1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; and 5,661,016; Marks et al., Bio/Technology 10: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851(1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg andHuszar, Intern. Rev. Immunol. 13: 65-93 (1995).

The term “naked antibody” refers to an antibody that is not conjugatedto a cytotoxic moiety or radiolabel.

The terms “full-length antibody,” “intact antibody” or “whole antibody”are used interchangeably to refer to an antibody in its substantiallyintact form, as opposed to an antibody fragment. Specifically wholeantibodies include those with heavy and light chains including an Fcregion. The constant domains may be native sequence constant domains(e.g., human native sequence constant domains) or amino acid sequencevariants thereof. In some cases, the intact antibody may have one ormore effector functions.

An “antibody fragment” comprises a portion of an intact antibody,preferably the antigen binding and/or the variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂ andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]);single-chain antibody molecules and multispecific antibodies formed fromantibody fragments. Papain digestion of antibodies produced twoidentical antigen-binding fragments, called “Fab” fragments, and aresidual “Fc” fragment, a designation reflecting the ability tocrystallize readily. The Fab fragment consists of an entire L chainalong with the variable region domain of the H chain (V_(H)), and thefirst constant domain of one heavy chain (C_(H)1). Each Fab fragment ismonovalent with respect to antigen binding, i.e., it has a singleantigen-binding site. Pepsin treatment of an antibody yields a singlelarge F(ab′)₂ fragment which roughly corresponds to two disulfide linkedFab fragments having different antigen-binding activity and is stillcapable of cross-linking antigen. Fab′ fragments differ from Fabfragments by having a few additional residues at the carboxy terminus ofthe C_(H)1 domain including one or more cysteines from the antibodyhinge region. Fab′-SH is the designation herein for Fab′ in which thecysteine residue(s) of the constant domains bear a free thiol group.F(ab′)₂ antibody fragments originally were produced as pairs of Fab′fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

The Fc fragment comprises the carboxy-terminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fc region, the region which is alsorecognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three HVRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the V_(H) and V_(L) antibody domains connectedinto a single polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding. For areview of the sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

“Functional fragments” of the antibodies of the invention comprise aportion of an intact antibody, generally including the antigen bindingor variable region of the intact antibody or the Fc region of anantibody which retains or has modified FcR binding capability. Examplesof antibody fragments include linear antibody, single-chain antibodymolecules and multispecific antibodies formed from antibody fragments.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10) residues) between the V_(H) and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,thereby resulting in a bivalent fragment, i.e., a fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the V_(H) and V_(L) domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described in greater detail in, for example, EP 404,097; WO93/11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448(1993).

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is(are) identical with or homologous to corresponding sequencesin antibodies derived from another species or belonging to anotherantibody class or subclass, as well as fragments of such antibodies, solong as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855(1984)). Chimeric antibodies of interest herein include PRIMATIZED®antibodies wherein the antigen-binding region of the antibody is derivedfrom an antibody produced by, e.g., immunizing macaque monkeys with anantigen of interest. As used herein, “humanized antibody” is used asubset of “chimeric antibodies.”

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from an HVR(hereinafter defined) of the recipient are replaced by residues from anHVR of a non-human species (donor antibody) such as mouse, rat, rabbitor non-human primate having the desired specificity, affinity, and/orcapacity. In some instances, framework (“FR”) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications may be made to further refine antibody performance, suchas binding affinity. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin sequence, and all orsubstantially all of the FR regions are those of a human immunoglobulinsequence, although the FR regions may include one or more individual FRresidue substitutions that improve antibody performance, such as bindingaffinity, isomerization, immunogenicity, etc. The number of these aminoacid substitutions in the FR are typically no more than 6 in the Hchain, and in the L chain, no more than 3. The humanized antibodyoptionally will also comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see, e.g., Jones et al., Nature 321:522-525 (1986);Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op.Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani andHamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris,Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr.Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and7,087,409.

A “human antibody” is an antibody that possesses an amino-acid sequencecorresponding to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art, including phage-display libraries. Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991). Also available for the preparation of human monoclonalantibodies are methods described in Cole et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J.Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel,Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can beprepared by administering the antigen to a transgenic animal that hasbeen modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled, e.g., immunizedxenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regardingXENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl.Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodiesgenerated via a human B-cell hybridoma technology.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). In native antibodies, H3 and L3 display the most diversityof the six HVRs, and H3 in particular is believed to play a unique rolein conferring fine specificity to antibodies. See, e.g., Xu et al.,Immunity 13:37-45 (2000); Johnson and Wu, in Methods in MolecularBiology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed,naturally occurring camelid antibodies consisting of a heavy chain onlyare functional and stable in the absence of light chain. See, e.g.,Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al.,Nature Struct. Biol. 3:733-736 (1996).

A number of HVR delineations are in use and are encompassed herein. TheKabat Complementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk, J.Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromisebetween the Kabat HVRs and Chothia structural loops, and are used byOxford Molecular's AbM antibody modeling software. The “contact” HVRsare based on an analysis of the available complex crystal structures.The residues from each of these HVRs are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1), 50-65 or49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variabledomain residues are numbered according to Kabat et al., supra, for eachof these definitions.

The expression “variable-domain residue-numbering as in Kabat” or“amino-acid-position numbering as in Kabat,” and variations thereof,refers to the numbering system used for heavy-chain variable domains orlight-chain variable domains of the compilation of antibodies in Kabatet al., supra. Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or HVR of the variable domain.For example, a heavy-chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g. residues 82a, 82b, and 82c, etc. according toKabat) after heavy-chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

“Framework” or “FR” residues are those variable-domain residues otherthan the HVR residues as herein defined.

A “human consensus framework” or “acceptor human framework” is aframework that represents the most commonly occurring amino acidresidues in a selection of human immunoglobulin VL or VH frameworksequences. Generally, the selection of human immunoglobulin VL or VHsequences is from a subgroup of variable domain sequences. Generally,the subgroup of sequences is a subgroup as in Kabat et al., Sequences ofProteins of Immunological Interest, 5^(th) Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991). Examples includefor the VL, the subgroup may be subgroup kappa I, kappa II, kappa III orkappa IV as in Kabat et al., supra. Additionally, for the VH, thesubgroup may be subgroup I, subgroup II, or subgroup III as in Kabat etal., supra. Alternatively, a human consensus framework can be derivedfrom the above in which particular residues, such as when a humanframework residue is selected based on its homology to the donorframework by aligning the donor framework sequence with a collection ofvarious human framework sequences. An acceptor human framework “derivedfrom” a human immunoglobulin framework or a human consensus frameworkmay comprise the same amino acid sequence thereof, or it may containpre-existing amino acid sequence changes. In some embodiments, thenumber of pre-existing amino acid changes are 10 or less, 9 or less, 8or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 orless.

A “VH subgroup III consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable heavy subgroup III ofKabat et al., supra. In one embodiment, the VH subgroup III consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences: EVQLVESGGGLVQPGGSLRLSCAAS (HC-FR1)(SEQID NO:25), WVRQAPGKGLEWV (HC-FR2), (SEQ ID NO:26),RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (HC-FR3, SEQ ID NO:27), WGQGTLVTVSA(HC-FR4), (SEQ ID NO:28).

A “VL kappa I consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable light kappa subgroupI of Kabat et al., supra. In one embodiment, the VH subgroup I consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences: DIQMTQSPSSLSASVGDRVTITC (LC-FR1) (SEQID NO:29), WYQQKPGKAPKLLIY (LC-FR2) (SEQ ID NO:30),GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (LC-FR3)(SEQ ID NO:31), FGQGTKVEIKR(LC-FR4)(SEQ ID NO:32).

An “amino-acid modification” at a specified position, e.g. of the Fcregion, refers to the substitution or deletion of the specified residue,or the insertion of at least one amino acid residue adjacent thespecified residue. Insertion “adjacent” to a specified residue meansinsertion within one to two residues thereof. The insertion may beN-terminal or C-terminal to the specified residue. The preferred aminoacid modification herein is a substitution.

An “affinity-matured” antibody is one with one or more alterations inone or more HVRs thereof that result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody that does notpossess those alteration(s). In one embodiment, an affinity-maturedantibody has nanomolar or even picomolar affinities for the targetantigen. Affinity-matured antibodies are produced by procedures known inthe art. For example, Marks et al., Bio/Technology 10:779-783 (1992)describes affinity maturation by VH- and VL-domain shuffling. Randommutagenesis of HVR and/or framework residues is described by, forexample: Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994);Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol.155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995);and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

As use herein, the term “specifically binds to” or is “specific for”refers to measurable and reproducible interactions such as bindingbetween a target and an antibody, which is determinative of the presenceof the target in the presence of a heterogeneous population of moleculesincluding biological molecules. For example, an antibody thatspecifically binds to a target (which can be an epitope) is an antibodythat binds this target with greater affinity, avidity, more readily,and/or with greater duration than it binds to other targets. In oneembodiment, the extent of binding of an antibody to an unrelated targetis less than about 10% of the binding of the antibody to the target asmeasured, e.g., by a radioimmunoassay (RIA). In certain embodiments, anantibody that specifically binds to a target has a dissociation constant(Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, or ≦0.1 nM. In certainembodiments, an antibody specifically binds to an epitope on a proteinthat is conserved among the protein from different species. In anotherembodiment, specific binding can include, but does not require exclusivebinding.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2 (including IgG2A and IgG2B), IgG-3, or IgG-4 subtypes, IgA(including IgA-1 and IgA-2), IgE, IgD or IgM. The Ig fusions preferablyinclude the substitution of a domain of a polypeptide or antibodydescribed herein in the place of at least one variable region within anIg molecule. In a particularly preferred embodiment, the immunoglobulinfusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3regions of an IgG1 molecule. For the production of immunoglobulinfusions see also U.S. Pat. No. 5,428,130 issued Jun. 27, 1995. Forexample, useful immunoadhesins as second medicaments useful forcombination therapy herein include polypeptides that comprise theextracellular or PD-1 binding portions of PD-L1 or PD-L2 or theextracellular or PD-L1 or PD-L2 binding portions of PD-1, fused to aconstant domain of an immunoglobulin sequence, such as a PD-L1 ECD—Fc, aPD-L2 ECD—Fc, and a PD-1 ECD—Fc, respectively. Immunoadhesincombinations of Ig Fc and ECD of cell surface receptors are sometimestermed soluble receptors.

A “fusion protein” and a “fusion polypeptide” refer to a polypeptidehaving two portions covalently linked together, where each of theportions is a polypeptide having a different property. The property maybe a biological property, such as activity in vitro or in vivo. Theproperty may also be simple chemical or physical property, such asbinding to a target molecule, catalysis of a reaction, etc. The twoportions may be linked directly by a single peptide bond or through apeptide linker but are in reading frame with each other.

A “PD-1 oligopeptide,” “PD-L1 oligopeptide,” or “PD-L2 oligopeptide” isan oligopeptide that binds, preferably specifically, to a PD-1, PD-L1 orPD-L2 negative costimulatory polypeptide, respectively, including areceptor, ligand or signaling component, respectively, as describedherein. Such oligopeptides may be chemically synthesized using knownoligopeptide synthesis methodology or may be prepared and purified usingrecombinant technology. Such oligopeptides are usually at least about 5amino acids in length, alternatively at least about 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100amino acids in length or more. Such oligopeptides may be identifiedusing well known techniques. In this regard, it is noted that techniquesfor screening oligopeptide libraries for oligopeptides that are capableof specifically binding to a polypeptide target are well known in theart (see, e.g., U.S. Pat. Nos. 5,556,762, 5,750,373, 4,708,871,4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT PublicationNos. WO 84/03506 and WO84/03564; Geysen et al., Proc. Natl. Acad. Sci.U.S.A., 81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci.U.S.A., 82:178-182 (1985); Geysen et al., in Synthetic Peptides asAntigens, 130-149 (1986); Geysen et al., J. Immunol. Meth., 102:259-274(1987); Schoofs et al., J. Immunol., 140:611-616 (1988), Cwirla, S. E.et al. Proc. Natl. Acad. Sci. USA, 87:6378 (1990); Lowman, H. B. et al.Biochemistry, 30:10832 (1991); Clackson, T. et al. Nature, 352: 624(1991); Marks, J. D. et al., J. Mol. Biol., 222:581 (1991); Kang, A. S.et al. Proc. Natl. Acad. Sci. USA, 88:8363 (1991), and Smith, G. P.,Current Opin. Biotechnol., 2:668 (1991).

A “blocking” antibody or an “antagonist” antibody is one that inhibitsor reduces a biological activity of the antigen it binds. In someembodiments, blocking antibodies or antagonist antibodies substantiallyor completely inhibit the biological activity of the antigen. Theanti-PD-L1 antibodies of the invention block the signaling through PD-1so as to restore a functional response by T-cells (e.g., proliferation,cytokine production, target cell killing) from a dysfunctional state toantigen stimulation.

An “agonist” or activating antibody is one that enhances or initiatessignaling by the antigen to which it binds. In some embodiments, agonistantibodies cause or activate signaling without the presence of thenatural ligand.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain, including native-sequence Fc regions andvariant Fc regions. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy-chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheC-terminal lysine (residue 447 according to the EU numbering system) ofthe Fc region may be removed, for example, during production orpurification of the antibody, or by recombinantly engineering thenucleic acid encoding a heavy chain of the antibody. Accordingly, acomposition of intact antibodies may comprise antibody populations withall K447 residues removed, antibody populations with no K447 residuesremoved, and antibody populations having a mixture of antibodies withand without the K447 residue. Suitable native-sequence Fc regions foruse in the antibodies of the invention include human IgG1, IgG2 (IgG2A,IgG2B), IgG3 and IgG4.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors, FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see M. Daëron, Annu.Rev. Immunol. 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet,Annu. Rev. Immunol. 9: 457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995).Other FcRs, including those to be identified in the future, areencompassed by the term “FcR” herein.

The term “Fc receptor” or “FcR” also includes the neonatal receptor,FcRn, which is responsible for the transfer of maternal IgGs to thefetus. Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J.Immunol. 24: 249 (1994). Methods of measuring binding to FcRn are known(see, e.g., Ghetie and Ward, Immunol. Today 18: (12): 592-8 (1997);Ghetie et al., Nature Biotechnology 15 (7): 637-40 (1997); Hinton etal., J. Biol. Chem. 279 (8): 6213-6 (2004); WO 2004/92219 (Hinton etal.). Binding to FcRn in vivo and serum half-life of human FcRnhigh-affinity binding polypeptides can be assayed, e.g., in transgenicmice or transfected human cell lines expressing human FcRn, or inprimates to which the polypeptides having a variant Fc region areadministered. WO 2004/42072 (Presta) describes antibody variants whichimproved or diminished binding to FcRs. See also, e.g., Shields et al.,J. Biol. Chem. 9(2): 6591-6604 (2001).

The phrase “substantially reduced,” or “substantially different,” asused herein, denotes a sufficiently high degree of difference betweentwo numeric values (generally one associated with a molecule and theother associated with a reference/comparator molecule) such that one ofskill in the art would consider the difference between the two values tobe of statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values). The differencebetween said two values is, for example, greater than about 10%, greaterthan about 20%, greater than about 30%, greater than about 40%, and/orgreater than about 50% as a function of the value for thereference/comparator molecule.

The term “substantially similar” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (for example, one associated with an antibody of theinvention and the other associated with a reference/comparatorantibody), such that one of skill in the art would consider thedifference between the two values to be of little or no biologicaland/or statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values). The differencebetween said two values is, for example, less than about 50%, less thanabout 40%, less than about 30%, less than about 20%, and/or less thanabout 10% as a function of the reference/comparator value.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers that are nontoxic to the cell or mammal beingexposed thereto at the dosages and concentrations employed. Often thephysiologically acceptable carrier is an aqueous pH buffered solution.Examples of physiologically acceptable carriers include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid; low molecular weight (less than about 10 residues)polypeptide; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol(PEG), and PLURONICS™.

A “package insert” refers to instructions customarily included incommercial packages of medicaments that contain information about theindications customarily included in commercial packages of medicamentsthat contain information about the indications, usage, dosage,administration, contraindications, other medicaments to be combined withthe packaged product, and/or warnings concerning the use of suchmedicaments, etc.

As used herein, the term “treatment” refers to clinical interventiondesigned to alter the natural course of the individual or cell beingtreated during the course of clinical pathology. Desirable effects oftreatment include decreasing the rate of disease progression,ameliorating or palliating the disease state, and remission or improvedprognosis. For example, an individual is successfully “treated” if oneor more symptoms associated with cancer are mitigated or eliminated,including, but are not limited to, reducing the proliferation of (ordestroying) cancerous cells, decreasing symptoms resulting from thedisease, increasing the quality of life of those suffering from thedisease, decreasing the dose of other medications required to treat thedisease, delaying the progression of the disease, and/or prolongingsurvival of individuals.

As used herein, “delaying progression of a disease” means to defer,hinder, slow, retard, stabilize, and/or postpone development of thedisease (such as cancer). This delay can be of varying lengths of time,depending on the history of the disease and/or individual being treated.As is evident to one skilled in the art, a sufficient or significantdelay can, in effect, encompass prevention, in that the individual doesnot develop the disease. For example, a late stage cancer, such asdevelopment of metastasis, may be delayed.

As used herein, “reducing or inhibiting cancer relapse” means to reduceor inhibit tumor or cancer relapse or tumor or cancer progression.

As used herein, “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Included in this definition are benign andmalignant cancers as well as dormant tumors or micrometastatses.Examples of cancer include but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, and leukemia. More particular examples of suchcancers include squamous cell cancer, lung cancer (including small-celllung cancer, non-small cell lung cancer, adenocarcinoma of the lung, andsquamous carcinoma of the lung), cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer (includinggastrointestinal cancer), pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), and Meigs' syndrome.

As used herein, “metastasis” is meant the spread of cancer from itsprimary site to other places in the body. Cancer cells can break awayfrom a primary tumor, penetrate into lymphatic and blood vessels,circulate through the bloodstream, and grow in a distant focus(metastasize) in normal tissues elsewhere in the body. Metastasis can belocal or distant. Metastasis is a sequential process, contingent ontumor cells breaking off from the primary tumor, traveling through thebloodstream, and stopping at a distant site. At the new site, the cellsestablish a blood supply and can grow to form a life-threatening mass.Both stimulatory and inhibitory molecular pathways within the tumor cellregulate this behavior, and interactions between the tumor cell and hostcells in the distant site are also significant.

An “effective amount” is at least the minimum concentration required toeffect a measurable improvement or prevention of a particular disorder.An effective amount herein may vary according to factors such as thedisease state, age, sex, and weight of the patient, and the ability ofthe antibody to elicit a desired response in the individual. Aneffective amount is also one in which any toxic or detrimental effectsof the treatment are outweighed by the therapeutically beneficialeffects. For prophylactic use, beneficial or desired results includeresults such as eliminating or reducing the risk, lessening theseverity, or delaying the onset of the disease, including biochemical,histological and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting duringdevelopment of the disease. For therapeutic use, beneficial or desiredresults include clinical results such as decreasing one or more symptomsresulting from the disease, increasing the quality of life of thosesuffering from the disease, decreasing the dose of other medicationsrequired to treat the disease, enhancing effect of another medicationsuch as via targeting, delaying the progression of the disease, and/orprolonging survival. In the case of cancer or tumor, an effective amountof the drug may have the effect in reducing the number of cancer cells;reducing the tumor size; inhibiting (i.e., slow to some extent ordesirably stop) cancer cell infiltration into peripheral organs; inhibit(i.e., slow to some extent and desirably stop) tumor metastasis;inhibiting to some extent tumor growth; and/or relieving to some extentone or more of the symptoms associated with the disorder. An effectiveamount can be administered in one or more administrations. For purposesof this invention, an effective amount of drug, compound, orpharmaceutical composition is an amount sufficient to accomplishprophylactic or therapeutic treatment either directly or indirectly. Asis understood in the clinical context, an effective amount of a drug,compound, or pharmaceutical composition may or may not be achieved inconjunction with another drug, compound, or pharmaceutical composition.Thus, an “effective amount” may be considered in the context ofadministering one or more therapeutic agents, and a single agent may beconsidered to be given in an effective amount if, in conjunction withone or more other agents, a desirable result may be or is achieved.

As used herein, “in conjunction with” refers to administration of onetreatment modality in addition to another treatment modality. As such,“in conjunction with” refers to administration of one treatment modalitybefore, during, or after administration of the other treatment modalityto the individual.

As used herein, “subject” is meant a mammal, including, but not limitedto, a human or non-human mammal, such as a bovine, equine, canine,ovine, or feline. Preferably, the subject is a human. Patients are alsosubjects herein.

As used herein, “complete response” or “CR” refers to disappearance ofall target lesions; “partial response” or “PR” refers to at least a 30%decrease in the sum of the longest diameters (SLD) of target lesions,taking as reference the baseline SLD; and “stable disease” or “SD”refers to neither sufficient shrinkage of target lesions to qualify forPR, nor sufficient increase to qualify for PD, taking as reference thesmallest SLD since the treatment started.

As used herein, “progressive disease” or “PD” refers to at least a 20%increase in the SLD of target lesions, taking as reference the smallestSLD recorded since the treatment started or the presence of one or morenew lesions.

As used herein, “progression free survival” (PFS) refers to the lengthof time during and after treatment during which the disease beingtreated (e.g., cancer) does not get worse. Progression-free survival mayinclude the amount of time patients have experienced a complete responseor a partial response, as well as the amount of time patients haveexperienced stable disease.

As used herein, “overall response rate” (ORR) refers to the sum ofcomplete response (CR) rate and partial response (PR) rate.

As used herein, “overall survival” refers to the percentage ofindividuals in a group who are likely to be alive after a particularduration of time.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®);alkyl sulfonates such as busulfan, improsulfan, and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinicacid; a camptothecin (including the synthetic analogue topotecan(HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin,scopolectin, and 9-aminocamptothecin); bryostatin; pemetrexed;callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesinsynthetic analogues); podophyllotoxin; podophyllinic acid; teniposide;cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; TLK-286; CDP323, an oral alpha-4integrin inhibitor; a sarcodictyin; spongistatin; nitrogen mustards suchas chlorambucil, chlornaphazine, cholophosphamide, estramustine,ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichin, phenesterine, prednimustine, trofosfamide,uracil mustard; nitrosureas such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such asthe enediyne antibiotics (e. g., calicheamicin, especially calicheamicingamma1I and calicheamicin omegaI1 (see, e.g., Nicolaou et al., Angew.Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(including ADRIAMYCIN®, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HClliposome injection (DOXIL®) and deoxydoxorubicin), epirubicin,esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C,mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin,puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin,tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such asmethotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine(XELODA®), an epothilone, and 5-fluorouracil (5-FU); folic acidanalogues such as denopterin, methotrexate, pteropterin, trimetrexate;purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine,thioguanine; pyrimidine analogs such as ancitabine, azacitidine,6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,enocitabine, floxuridine, and imatinib (a 2-phenylaminopyrimidinederivative), as well as other c-Kit inhibitors; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate;etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine;maytansinoids such as maytansine and ansamitocins; mitoguazone;mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet;pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK®polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane;rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®,FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol;pipobroman; gacytosine; arabinoside (“Ara-C”); thiotepa; taxoids, e.g.,paclitaxel (TAXOL®), albumin-engineered nanoparticle formulation ofpaclitaxel (ABRAXANE™), and doxetaxel (TAXOTERE®); chloranbucil;6-thioguanine; mercaptopurine; methotrexate; platinum analogs such ascisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide(VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin;leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate;daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000;difluorometlhylornithine (DMFO); retinoids such as retinoic acid;pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovovin.

Also included in this definition are anti-hormonal agents that act toregulate, reduce, block, or inhibit the effects of hormones that canpromote the growth of cancer, and are often in the form of systemic, orwhole-body treatment. They may be hormones themselves. Examples includeanti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including NOLVADEX® tamoxifen),raloxifene (EVISTA®), droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY117018, onapristone, and toremifene (FARESTON®);anti-progesterones; estrogen receptor down-regulators (ERDs); estrogenreceptor antagonists such as fulvestrant (FASLODEX®); agents thatfunction to suppress or shut down the ovaries, for example, leutinizinghormone-releasing hormone (LHRH) agonists such as leuprolide acetate(LUPRON® and ELIGARD®), goserelin acetate, buserelin acetate andtripterelin; anti-androgens such as flutamide, nilutamide andbicalutamide; and aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrolacetate (MEGASE®), exemestane (AROMASIN®), formestanie, fadrozole,vorozole (RIVISOR®), letrozole (FEMARA®), and anastrozole (ARIMIDEX®).In addition, such definition of chemotherapeutic agents includesbisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®),etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®),alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), orrisedronate (ACTONEL®); as well as troxacitabine (a 1,3-dioxolanenucleoside cytosine analog); anti-sense oligonucleotides, particularlythose that inhibit expression of genes in signaling pathways implicatedin abherant cell proliferation, such as, for example, PKC-alpha, Raf,H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such asTHERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN®vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1inhibitor (e.g., LURTOTECAN®); an anti-estrogen such as fulvestrant; aKit inhibitor such as imatinib or EXEL-0862 (a tyrosine kinaseinhibitor); EGFR inhibitor such as erlotinib or cetuximab; an anti-VEGFinhibitor such as bevacizumab; arinotecan; rmRH (e.g., ABARELIX®);lapatinib and lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosinekinase small-molecule inhibitor also known as GW572016); 17AAG(geldanamycin derivative that is a heat shock protein (Hsp) 90 poison),and pharmaceutically acceptable salts, acids or derivatives of any ofthe above.

As used herein, the term “cytokine” refers generically to proteinsreleased by one cell population that act on another cell asintercellular mediators or have an autocrine effect on the cellsproducing the proteins. Examples of such cytokines include lymphokines,monokines; interleukins (“ILs”) such as IL-1, IL-1α, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL10, IL-11, IL-12, IL-13, IL-15,IL-17A-F, IL-18 to IL-29 (such as IL-23), IL-31, including PROLEUKIN®rIL-2; a tumor-necrosis factor such as TNF-α or TNF-β, TGF-β1-3; andother polypeptide factors including leukemia inhibitory factor (“LIF”),ciliary neurotrophic factor (“CNTF”), CNTF-like cytokine (“CLC”),cardiotrophin (“CT”), and kit ligand (“KL”).

As used herein, the term “chemokine” refers to soluble factors (e.g.,cytokines) that have the ability to selectively induce chemotaxis andactivation of leukocytes. They also trigger processes of angiogenesis,inflammation, wound healing, and tumorigenesis. Example chemokinesinclude IL-8, a human homolog of murine keratinocyte chemoattractant(KC).

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

The phrase “pharmaceutically acceptable salt” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a compound ofthe invention. Exemplary salts include, but are not limited, to sulfate,citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucuronate, saccharate, formate, benzoate, glutamate,methanesulfonate “mesylate”, ethanesulfonate, benzenesulfonate,p-toluenesulfonate, pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g.,sodium and potassium) salts, alkaline earth metal (e.g., magnesium)salts, and ammonium salts. A pharmaceutically acceptable salt mayinvolve the inclusion of another molecule such as an acetate ion, asuccinate ion or other counter ion. The counter ion may be any organicor inorganic moiety that stabilizes the charge on the parent compound.Furthermore, a pharmaceutically acceptable salt may have more than onecharged atom in its structure. Instances where multiple charged atomsare part of the pharmaceutically acceptable salt can have multiplecounter ions. Hence, a pharmaceutically acceptable salt can have one ormore charged atoms and/or one or more counter ion.

If the compound of the invention is a base, the desired pharmaceuticallyacceptable salt may be prepared by any suitable method available in theart, for example, treatment of the free base with an inorganic acid,such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,methanesulfonic acid, phosphoric acid and the like, or with an organicacid, such as acetic acid, maleic acid, succinic acid, mandelic acid,fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid,salicylic acid, a pyranosidyl acid, such as glucuronic acid orgalacturonic acid, an alpha hydroxy acid, such as citric acid ortartaric acid, an amino acid, such as aspartic acid or glutamic acid, anaromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid,such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.

If the compound of the invention is an acid, the desiredpharmaceutically acceptable salt may be prepared by any suitable method,for example, treatment of the free acid with an inorganic or organicbase, such as an amine (primary, secondary or tertiary), an alkali metalhydroxide or alkaline earth metal hydroxide, or the like. Illustrativeexamples of suitable salts include, but are not limited to, organicsalts derived from amino acids, such as glycine and arginine, ammonia,primary, secondary, and tertiary amines, and cyclic amines, such aspiperidine, morpholine and piperazine, and inorganic salts derived fromsodium, calcium, potassium, magnesium, manganese, iron, copper, zinc,aluminum and lithium.

The phrase “pharmaceutically acceptable” indicates that the substance orcomposition must be compatible chemically and/or toxicologically, withthe other ingredients comprising a formulation, and/or the mammal beingtreated therewith.

It is understood that aspects and variations of the invention describedherein include “consisting” and/or “consisting essentially of” aspectsand variations.

III. Methods

In one aspect, provided herein is a method for treating or delayingprogression of cancer in an individual comprising administering to theindividual an effective amount of a PD-1 axis binding antagonist incombination with an agent that decreases or inhibits TIGIT expressionand/or activity.

In another aspect, provided herein is a method for reducing orinhibiting cancer relapse or cancer progression in an individualcomprising administering to the individual an effect amount of a PD-1axis binding antagonist in combination with an agent that that decreasesor inhibits TIGIT expression and/or activity. As disclosed herein,cancer relapse and/or cancer progression include, without limitation,cancer metastasis.

In another aspect, provided herein is a method for treating or delayingprogression of an immune related disease in an individual comprisingadministering to the individual an effect amount of a PD-1 axis bindingantagonist in combination with an agent that that decreases or inhibitsTIGIT expression and/or activity.

In another aspect, provided herein is a method for reducing orinhibiting progression of an immune related disease in an individualcomprising administering to the individual an effect amount of a PD-1axis binding antagonist in combination with an agent that that decreasesor inhibits TIGIT expression and/or activity.

In some embodiments, the immune related disease is associated with Tcell dysfunctional disorder. In some embodiments, the immune relateddisease is a viral infection. In certain embodiments, the viralinfection is a chronic viral infection. In some embodiments, T celldysfunctional disorder is characterized by decreased responsiveness toantigenic stimulation. In some embodiments, the T cell dysfunctionaldisorder is characterized by T cell anergy or decreased ability tosecrete cytokines, proliferate or execute cytolytic activity. In someembodiments, the T cell dysfunctional disorder is characterized by Tcell exhaustion. In some embodiments, the T cells are CD4+ and CD8+ Tcells. In some embodiments, the T cell dysfunctional disorder includesunresolved acute infection, chronic infection and tumor immunity.

In another aspect, provided herein is a method for increasing, enhancingor stimulating an immune response or function in an individualcomprising administering to the individual an effect amount of a PD-1axis binding antagonist in combination with an agent that decreases orinhibits TIGIT expression and/or activity.

In another aspect, provided herein is a method of treating or delayingprogression of cancer in an individual comprising administering to theindividual an effective amount of a PD-1 axis binding antagonist and anagent that modulates the CD226 expression and/or activity.

In another aspect, provided herein is a method for reducing orinhibiting cancer relapse or cancer progression in an individualcomprising administering to the individual an effective amount of a PD-1axis binding antagonist and an agent that modulates the CD226 expressionand/or activity.

In another aspect, provided herein is a method for treating or delayingprogression of an immune related disease in an individual comprisingadministering to the individual an effective amount of a PD-1 axisbinding antagonist and an agent that modulates the CD226 expressionand/or activity.

In another aspect, provided herein is a method for reducing orinhibiting progression of an immune related disease in an individualcomprising administering to the individual an effective amount of a PD-1axis binding antagonist and agent that modulates the CD226 expressionand/or activity.

In some embodiments, the immune related disease is associated with Tcell dysfunctional disorder. In some embodiments, the immune relateddisease is a viral infection. In certain embodiments, the viralinfection is a chronic viral infection. In some embodiments, the T celldysfunctional disorder is characterized by decreased responsiveness toantigenic stimulation. In some embodiments, the T cell dysfunctionaldisorder is characterized by T cell anergy, or decreased ability tosecrete cytokines, proliferate or execute cytolytic activity. In someembodiments, the T cell dysfunctional disorder is characterized by Tcell exhaustion. In some embodiments, the T cells are CD4+ and CD8+ Tcells. In some embodiments, the immune related disease is selected fromthe group consisting of unresolved acute infection, chronic infectionand tumor immunity.

In another aspect, provided herein is a method of increasing, enhancingor stimulating an immune response or function in an individual byadministering to the individual an effective amount of a PD-1 axisbinding antagonist and an agent that modulates the CD226 expressionand/or activity.

In some embodiments, the agent that modulates the CD226 expressionand/or activity is capable of increasing and/or stimulating CD226expression and/or activity; increasing and/or stimulating theinteraction of CD226 with PVR, PVRL2, and/or PVRL3; and increasingand/or stimulating the intracellular signaling mediated by CD226 bindingto PVR, PVRL2, and/or PVRL3. As used herein, an agent that is capable ofincreasing and/or stimulating CD226 expression and/or activity includes,without limitation, agents that increase and/or stimulate CD226expression and/or activity. As used herein, an agent that is capable ofincreasing and/or stimulating the interaction of CD226 with PVR, PVRL2,and/or PVRL3 includes, without limitation, agents that increase and/orstimulate the interaction of CD226 with PVR, PVRL2, and/or PVRL3. Asused herein, an agent that is capable of increasing and/or stimulatingthe intracellular signaling mediated by CD226 binding to PVR, PVRL2,and/or PVRL3 includes, without limitation, agents that increase and/orstimulate the intracellular signaling mediated by CD226 binding to PVR,PVRL2, and/or PVRL3.

In some embodiments, the agent that modulates the CD226 expressionand/or activity is selected from an agent that inhibits and/or blocksthe interaction of CD226 with TIGIT, an antagonist of TIGIT expressionand/or activity, an antagonist of PVR expression and/or activity, anagent that inhibits and/or blocks the interaction of TIGIT with PVR, anagent that inhibits and/or blocks the interaction of TIGIT with PVRL2,an agent that inhibits and/or blocks the interaction of TIGIT withPVRL3, an agent that inhibits and/or blocks the intracellular signalingmediated by TIGIT binding to PVR, an agent that inhibits and/or blocksthe intracellular signaling mediated by TIGIT binding to PVRL2, an agentthat inhibits and/or blocks the intracellular signaling mediated byTIGIT binding to PVRL3, and combinations thereof.

In some embodiments, the agent that inhibits and/or blocks theinteraction of CD226 with TIGIT is a small molecule inhibitor, aninhibitory antibody or antigen-binding fragment thereof, an aptamer, aninhibitory nucleic acid, and an inhibitory polypeptide. In someembodiments, the agent that inhibits and/or blocks the interaction ofCD226 with TIGIT is an anti-TIGIT antibody or antigen-binding fragmentthereof. In some embodiments, the agent that inhibits and/or blocks theinteraction of CD226 with TIGIT is an inhibitory nucleic acid selectedfrom an antisense polynucleotide, an interfering RNA, a catalytic RNA,and an RNA-DNA chimera.

In some embodiments, the antagonist of TIGIT expression and/or activityis a small molecule inhibitor, an inhibitory antibody or antigen-bindingfragment thereof, an aptamer, an inhibitory nucleic acid, and aninhibitory polypeptide. In some embodiments, the antagonist of TIGITexpression and/or activity is an anti-TIGIT antibody or antigen-bindingfragment thereof. In some embodiments, the antagonist of TIGITexpression and/or activity is an inhibitory nucleic acid selected froman antisense polynucleotide, an interfering RNA, a catalytic RNA, and anRNA-DNA chimera.

In some embodiments, the antagonist of PVR expression and/or activity isa small molecule inhibitor, an inhibitory antibody or antigen-bindingfragment thereof, an aptamer, an inhibitory nucleic acid, and aninhibitory polypeptide. In some embodiments, the antagonist of PVRexpression and/or activity is selected from a small molecule inhibitor,an inhibitory antibody or antigen-binding fragment thereof, an aptamer,an inhibitory nucleic acid, and an inhibitory polypeptide.

In some embodiments, the agent that inhibits and/or blocks theinteraction of TIGIT with PVR is a small molecule inhibitor, aninhibitory antibody or antigen-binding fragment thereof, an aptamer, aninhibitory nucleic acid, and an inhibitory polypeptide. In someembodiments, the agent that inhibits and/or blocks the interaction ofTIGIT with PVR is selected from a small molecule inhibitor, aninhibitory antibody or antigen-binding fragment thereof, an aptamer, aninhibitory nucleic acid, and an inhibitory polypeptide.

In some embodiments, the agent that inhibits and/or blocks theinteraction of TIGIT with PVRL2 is selected from a small moleculeinhibitor, an inhibitory antibody or antigen-binding fragment thereof,an aptamer, an inhibitory nucleic acid, and an inhibitory polypeptide.

In some embodiments, the agent that inhibits and/or blocks theinteraction of TIGIT with PVRL3 is selected from a small moleculeinhibitor, an inhibitory antibody or antigen-binding fragment thereof,an aptamer, an inhibitory nucleic acid, and an inhibitory polypeptide.

In some embodiments, the agent that inhibits and/or blocks theintracellular signaling mediated by TIGIT binding to PVR is a smallmolecule inhibitor, an inhibitory antibody or antigen-binding fragmentthereof, an aptamer, an inhibitory nucleic acid, and an inhibitorypolypeptide. In some embodiments, the agent that inhibits and/or blocksthe intracellular signaling mediated by TIGIT binding to PVR is selectedfrom a small molecule inhibitor, an inhibitory antibody orantigen-binding fragment thereof, an aptamer, an inhibitory nucleicacid, and an inhibitory polypeptide.

In some embodiments, the agent that inhibits and/or blocks theintracellular signaling mediated by TIGIT binding to PVRL2 is selectedfrom a small molecule inhibitor, an inhibitory antibody orantigen-binding fragment thereof, an aptamer, an inhibitory nucleicacid, and an inhibitory polypeptide.

In some embodiments, the agent that inhibits and/or blocks theintracellular signaling mediated by TIGIT binding to PVRL3 is selectedfrom a small molecule inhibitor, an inhibitory antibody orantigen-binding fragment thereof, an aptamer, an inhibitory nucleicacid, and an inhibitory polypeptide.

In another aspect, provided herein is a method of increasing, enhancingor stimulating an immune response or function in an individual byadministering to the individual an effective amount of an agent thatdecreases or inhibits TIGIT expression and/or activity and an agent thatdecreases or inhibits the expression and/or activity of one or moreadditional immune co-inhibitory receptors. In some embodiments, the oneof more additional immune co-inhibitory receptor is selected from PD-1,CTLA-4, LAG3, TIM3, BTLA VISTA, B7H4, and CD96. In some embodiments, oneof more additional immune co-inhibitory receptor is selected from PD-1,CTLA-4, LAG3 and TIM3.

In another aspect, provided herein is a method of increasing, enhancingor stimulating an immune response or function in an individual byadministering to the individual an effective amount of an agent thatdecreases or inhibits TIGIT expression and/or activity and an agent thatincreases or activates the expression and/or activity of one or moreadditional immune co-stimulatory receptors. In some embodiments, the oneof more additional immune co-stimulatory receptor is selected fromCD226, OX-40, CD28, CD27, CD137, HVEM, GITR, MICA, ICOS, NKG2D, and 2B4.In some embodiments, the one or more additional immune co-stimulatoryreceptor is selected from CD226, OX-40, CD28, CD27, CD137, HVEM, andGITR. In some embodiments, the one of more additional immuneco-stimulatory receptor is selected from OX-40 and CD27.

The methods of this invention may find use in treating conditions whereenhanced immunogenicity is desired such as increasing tumorimmunogenicity for the treatment of cancer or T cell dysfunctionaldisorders.

A variety of cancers may be treated, or their progression may bedelayed.

In some embodiments, the individual has non-small cell lung cancer. Thenon-small cell lung cancer may be at early stage or at late stage. Insome embodiments, the individual has small cell lung cancer. The smallcell lung cancer may be at early stage or at late stage. In someembodiments, the individual has renal cell cancer. The renal cell cancermay be at early stage or at late stage. In some embodiments, theindividual has colorectal cancer. The colorectal cancer may be at earlystage or late stage. In some embodiments, the individual has ovariancancer. The ovarian cancer may be at early stage or at late stage. Insome embodiments, the individual has breast cancer. The breast cancermay be at early stage or at late stage. In some embodiments, theindividual has pancreatic cancer. The pancreatic cancer may be at earlystage or at late stage. In some embodiments, the individual has gastriccarcinoma. The gastric carcinoma may be at early stage or at late stage.In some embodiments, the individual has bladder cancer. The bladdercancer may be at early stage or at late stage. In some embodiments, theindividual has esophageal cancer. The esophageal cancer may be at earlystage or at late stage. In some embodiments, the individual hasmesothelioma. The mesothelioma may be at early stage or at late stage.In some embodiments, the individual has melanoma. The melanoma may be atearly stage or at late stage. In some embodiments, the individual hashead and neck cancer. The head and neck cancer may be at early stage orat late stage. In some embodiments, the individual has thyroid cancer.The thyroid cancer may be at early stage or at late stage. In someembodiments, the individual has sarcoma. The sarcoma may be at earlystage or late stage. In some embodiments, the individual has prostatecancer. The prostate cancer may be at early stage or at late stage. Insome embodiments, the individual has glioblastoma. The glioblastoma maybe at early stage or at late stage. In some embodiments, the individualhas cervical cancer. The cervical cancer may be at early stage or atlate stage. In some embodiments, the individual has thymic carcinoma.The thymic carcinoma may be at early stage or at late stage. In someembodiments, the individual has leukemia. The leukemia may be at earlystage or at late stage. In some embodiments, the individual haslymphomas. The lymphoma may be at early stage or at late stage. In someembodiments, the individual has myelomas. The myelomas may be at earlystage or at late stage. In some embodiments, the individual has mycosesfungoids. The mycoses fungoids may be at early stage or at late stage.In some embodiments, the individual has merkel cell cancer. The merkelcell cancer may be at early stage or at late stage. In some embodiments,the individual has hematologic malignancies. The hematologicalmalignancies may be early stage or late stage. In some embodiments, theindividual is a human.

In some embodiments of the methods of this invention, the CD4 and/or CD8T cells in the individual have increased or enhanced priming,activation, proliferation, cytokine release and/or cytolytic activityrelative to prior to the administration of the combination.

In some embodiments of the methods of this invention, the number of CD4and/or CD8 T cells is elevated relative to prior to administration ofthe combination. In some embodiments of the methods of this invention,the number of activated CD4 and/or CD8 T cells is elevated relative toprior to administration of the combination.

In some embodiments of the methods of this invention, the activated CD4and/or CD8 T cells is characterized by γ-IFN⁺ producing CD4 and/or CD8 Tcells and/or enhanced cytolytic activity relative to prior to theadministration of the combination.

In some embodiments of the methods of this invention, the CD4 and/or CD8T cells exhibit increased release of cytokines selected from the groupconsisting of IFN-γ, TNF-α, and interleukins.

In some embodiments of the methods of this invention, the CD4 and/or CD8T cell is an effector memory T cell. In some embodiments of the methodsof this invention, the CD4 and/or CD8 effector memory T cell ischaracterized by γ-IFN⁺ producing CD4 and/or CD8 T cells and/or enhancedcytolytic activity. In some embodiments of the methods of thisinvention, the CD4 and/or CD8 effector memory T cell is characterized byhaving the expression of CD44^(high) CD62L^(low).

In some embodiments of the methods of this invention, the cancer haselevated levels of T cell infiltration.

In some embodiments, the methods of the invention may further compriseadministering an additional therapy. The additional therapy may beradiation therapy, surgery, chemotherapy, gene therapy, DNA therapy,viral therapy, RNA therapy, immunotherapy, bone marrow transplantation,nanotherapy, monoclonal antibody therapy, or a combination of theforegoing. The additional therapy may be in the form of an adjuvant orneoadjuvant therapy. In some embodiments, the additional therapy is theadministration of side-effect limiting agents (e.g., agents intended tolessen the occurrence and/or severity of side effects of treatment, suchas anti-nausea agents, etc.). In some embodiments, the additionaltherapy is radiation therapy. In some embodiments, the additionaltherapy is surgery. In some embodiments, the additional therapy may beone or more of the chemotherapeutic agents described hereinabove.

Any of the PD-1 axis binding antagonists and agents that decreases orinhibits TIGIT expression and/or activity described below may be used inthe methods of the invention.

In some embodiments, any of the targets described herein (e.g., PD-1,PD-L1, PD-L2, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, CD96, B7-1, TIGIT,CD226, OX-40, CD28, CD27, CD137, HVEM, GITR, MICA, ICOS, NKG2D, 2B4,etc.) is a human protein.

PD-1 Axis Binding Antagonists

Provided herein is a method for treatment or delaying progression ofcancer in an individual comprising administering to the individual aneffective amount of a PD-1 axis binding antagonist in combination withan agent that decreases or inhibits TIGIT expression and/or activity.Provided herein is also a method for reducing or inhibiting cancerrelapse or cancer progression in an individual comprising administeringto the individual an effect amount of a PD-1 axis binding antagonist incombination with an agent that that decreases or inhibits TIGITexpression and/or activity. Provided herein is also a method fortreating or delaying progression of an immune related disease in anindividual comprising administering to the individual an effect amountof a PD-1 axis binding antagonist in combination with an agent that thatdecreases or inhibits TIGIT expression and/or activity. Provided hereinis also a method for reducing or inhibiting progression of an immunerelated disease in an individual comprising administering to theindividual an effect amount of a PD-1 axis binding antagonist incombination with an agent that that decreases or inhibits TIGITexpression and/or activity. Provided herein is also a method forincreasing, enhancing or stimulating an immune response or function inan individual comprising administering to the individual an effectamount of a PD-1 axis binding antagonist in combination with an agentthat decreases or inhibits TIGIT expression and/or activity.

For example, a PD-1 axis binding antagonist includes a PD-1 bindingantagonist, a PD-L1 binding antagonist and a PD-L2 binding antagonist.

In some embodiments, the PD-1 binding antagonist is a molecule thatinhibits the binding of PD-1 to its ligand binding partners. In aspecific aspect the PD-1 ligand binding partners are PD-L1 and/or PD-L2.In another embodiment, a PD-L1 binding antagonist is a molecule thatinhibits the binding of PD-L1 to its binding partners. In a specificaspect, PD-L1 binding partners are PD-1 and/or B7-1. In anotherembodiment, the PD-L2 binding antagonist is a molecule that inhibits thebinding of PD-L2 to its binding partners. In a specific aspect, a PD-L2binding partner is PD-1. The antagonist may be an antibody, an antigenbinding fragment thereof, an immunoadhesin, a fusion protein, oroligopeptide.

In some embodiments, the PD-1 binding antagonist is selected fromMDX-1106 (nivolumab), Merck 3745 (lambrolizumab), CT-011 (pidilizumab),and AMP-224. In some embodiments, the PD-L1 binding antagonist isselected from YW243.55.S70, MPDL3280A, MDX-1105, and MEDI 4736. In someembodiments, the PD-L2 binding antagonist is AMP-224. In someembodiments, the PD-1 binding antagonist is AMP-224. MDX-1105, alsoknown as BMS-936559, is an anti-PD-L1 antibody described inWO2007/005874. Antibody YW243.55.S70 (SEQ ID No. 20) is an anti-PD-L1described in WO 2010/077634 A1 and U.S. Pat. No. 8,217,149, which areincorporated herein by reference. MDX-1106, also known as MDX-1106-04,ONO-4538, BMS-936558, or nivolumab, is an anti-PD-1 antibody describedin WO2006/121168. Merck 3745, also known as MK 3475, MK-3475,SCH-900475, or lambrolizumab, is an anti-PD-1 antibody described inWO2009/114335. CT-011, also known as hBAT, hBAT-1, or pidilizumab, is ananti-PD-1 antibody described in WO2009/101611. AMP-224, also known asB7-DCIg, is a PD-L2-Fc fusion soluble receptor described inWO2010/027827 and WO2011/066342.

Examples of anti-PD-L1 antibodies useful for the methods of thisinvention, and methods for making thereof are described in PCT patentapplication WO 2010/077634 A1 and U.S. Pat. No. 8,217,149, which areincorporated herein by reference.

In some embodiments, the PD-1 axis binding antagonist is an anti-PD-L1antibody. In some embodiments, the anti-PD-L1 antibody is capable ofinhibiting binding between PD-L1 and PD-1 and/or between PD-L1 and B7-1.In some embodiments, the anti-PD-L1 antibody is a monoclonal antibody.In some embodiments, the anti-PD-L1 antibody is an antibody fragmentselected from the group consisting of Fab, Fab′-SH, Fv, scFv, and(Fab′)₂ fragments. In some embodiments, the anti-PD-L1 antibody is ahumanized antibody. In some embodiments, the anti-PD-L1 antibody is ahuman antibody.

The anti-PD-L1 antibodies useful in this invention, includingcompositions containing such antibodies, such as those described in WO2010/077634 A1 and U.S. Pat. No. 8,217,149, may be used in combinationwith an agent that decreases or inhibits TIGIT expression and/oractivity with or without any additional therapy (e.g., chemotherapy) totreat cancer or an immune related disease (e.g., T cell dysfunctionaldisorder, viral infection, chronic viral infection, etc.).

In one embodiment, the anti-PD-L1 antibody contains a heavy chainvariable region polypeptide comprising an HVR-H1, HVR-H2 and HVR-H3sequence, wherein:

(a) the HVR-H1 sequence is (SEQ ID NO: 33) GFTFSX₁SWIH;(b) the HVR-H2 sequence is (SEQ ID NO: 34) AWIX₂PYGGSX₃YYADSVKG;(c) the HVR-H3 sequence is (SEQ ID NO: 19) RHWPGGFDY;

further wherein: X₁ is D or G; X₂ is S or L; X₃ is T or S.

In one specific aspect, X₁ is D; X₂ is S and X₃ is T. In another aspect,the polypeptide further comprises variable region heavy chain frameworksequences juxtaposed between the HVRs according to the formula:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4). In yetanother aspect, the framework sequences are derived from human consensusframework sequences. In a further aspect, the framework sequences are VHsubgroup III consensus framework. In a still further aspect, at leastone of the framework sequences is the following:

HC-FR1 is (SEQ ID NO: 25) EVQLVESGGGLVQPGGSLRLSCAAS HC-FR2 is(SEQ ID NO: 26) WVRQAPGKGLEWV HC-FR3 is (SEQ ID NO: 27)RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR  HC-FR4 is (SEQ ID NO: 28) WGQGTLVTVSA.

In a still further aspect, the heavy chain polypeptide is furthercombined with a variable region light chain comprising an HVR-L1, HVR-L2and HVR-L3, wherein:

(a) the HVR-L1 sequence is (SEQ ID NO: 35) RASQX₄X₅X₆TX₇X₈A;(b) the HVR-L2 sequence is (SEQ ID NO: 36) SASX₉LX₁₀S,;(c) the HVR-L3 sequence is (SEQ ID NO: 37) QQX₁₁X₁₂X₁₃X₁₄PX₁₅T;

-   -   further wherein: X₄ is D or V; X₅ is V or I; X₆ is S or N; X₇ is        A or F; X₈ is V or L; X₉ is F or T; X₁₀ is Y or A; X₁₁ is Y, G,        F, or S; X₁₂ is L, Y, F or W; X₁₃ is Y, N, A, T, G, F or I; X₁₄        is H, V, P, T or I; X₁₅ is A, W, R, P or T.

In a still further aspect, X4 is D; X5 is V; X6 is S; X7 is A; X8 is V;X9 is F; X10 is Y; X11 is Y; X12 is L; X13 is Y; X14 is H; X15 is A. Ina still further aspect, the light chain further comprises variableregion light chain framework sequences juxtaposed between the HVRsaccording to the formula:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In astill further aspect, the framework sequences are derived from humanconsensus framework sequences. In a still further aspect, the frameworksequences are VL kappa I consensus framework. In a still further aspect,at least one of the framework sequence is the following:

LC-FR1 is (SEQ ID NO: 29) DIQMTQSPSSLSASVGDRVTITC LC-FR2 is(SEQ ID NO: 30) WYQQKPGKAPKLLIY LC-FR3 is (SEQ ID NO: 31)GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LC-FR4 is (SEQ ID NO: 32) FGQGTKVEIKR.

In another embodiment, provided is an isolated anti-PD-L1 antibody orantigen binding fragment comprising a heavy chain and a light chainvariable region sequence, wherein:

(a) the heavy chain comprises and HVR-H1, HVR-H2 and HVR-H3, whereinfurther:

(i) the HVR-H1 sequence is (SEQ ID NO: 33) GFTFSX₁SWIH;(ii) the HVR-H2 sequence is (SEQ ID NO: 34) AWIX₂PYGGSX₃YYADSVKG(iii) the HVR-H3 sequence is (SEQ ID NO: 19) RHWPGGFDY, and

(b) the light chain comprises and HVR-L1, HVR-L2 and HVR-L3, whereinfurther:

(i) the HVR-L1 sequence is (SEQ ID NO: 35) RASQX₄X₅X₆TX₇X₈A(ii) the HVR-L2 sequence is (SEQ ID NO: 36) SASX₉LX₁₀S; and(iii) the HVR-L3 sequence is (SEQ ID NO: 37) QQX₁₁X₁₂X₁₃X₁₄PX₁₅T;

-   -   further wherein: X₁ is D or G; X₂ is S or L; X₃ is T or S; X₄ is        D or V; X₅ is V or I; X₆ is S or N; X₇ is A or F; X₈ is V or L;        X₉ is F or T; X₁₀ is Y or A; X₁₁ is Y, G, F, or S; X₁₂ is L, Y,        F or W; X₁₃ is Y, N, A, T, G, F or I; X₁₄ is H, V, P, T or I;        X₁₅ is A, W, R, P or T.

In a specific aspect, X₁ is D; X₂ is S and X₃ is T. In another aspect,X₄ is D; X₅ is V; X₆ is S; X₇ is A; X₈ is V; X₉ is F; X₁₀ is Y; X₁₁ isY; X₁₂ is L; X₁₃ is Y; X₁₄ is H; X₁₅ is A. In yet another aspect, X₁ isD; X₂ is S and X₃ is T, X₄ is D; X₅ is V; X₆ is S; X₇ is A; X₈ is V; X₉is F; X₁₀ is Y; X₁₁ is Y; X₁₂ is L; X₁₃ is Y; X₁₄ is H and X₁₅ is A.

In a further aspect, the heavy chain variable region comprises one ormore framework sequences juxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In astill further aspect, the framework sequences are derived from humanconsensus framework sequences. In a still further aspect, the heavychain framework sequences are derived from a Kabat subgroup I, II, orIII sequence. In a still further aspect, the heavy chain frameworksequence is a VH subgroup III consensus framework. In a still furtheraspect, one or more of the heavy chain framework sequences is thefollowing:

HC-FR1 (SEQ ID NO: 25) EVQLVESGGGLVQPGGSLRLSCAAS HC-FR2 (SEQ ID NO: 26)WVRQAPGKGLEWV HC-FR3 (SEQ ID NO: 27) RFTISADTSKNTAYLQMNSLRAEDTAVYYCARHC-FR4 (SEQ ID NO: 28) WGQGTLVTVSA.

In a still further aspect, the light chain framework sequences arederived from a Kabat kappa I, II, II or IV subgroup sequence. In a stillfurther aspect, the light chain framework sequences are VL kappa Iconsensus framework. In a still further aspect, one or more of the lightchain framework sequences is the following:

LC-FR1 (SEQ ID NO: 29) DIQMTQSPSSLSASVGDRVTITC LC-FR2 (SEQ ID NO: 30)WYQQKPGKAPKLLIY LC-FR3 (SEQ ID NO: 31) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLC-FR4 (SEQ ID NO: 32) FGQGTKVEIKR.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG1. In a still further aspect, the murine constant region isselected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region if IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect the minimal effectorfunction results from an “effector-less Fc mutation” or aglycosylation.In still a further embodiment, the effector-less Fc mutation is an N297Aor D265A/N297A substitution in the constant region.

In yet another embodiment, provided is an anti-PD-L1 antibody comprisinga heavy chain and a light chain variable region sequence, wherein:

-   -   (a) the heavy chain further comprises and HVR-H1, HVR-H2 and an        HVR-H3 sequence having at least 85% sequence identity to        GFTFSDSWIH (SEQ ID NO:17), AWISPYGGSTYYADSVKG (SEQ ID NO:18) and        RHWPGGFDY (SEQ ID NO:19), respectively, or    -   (b) the light chain further comprises an HVR-L1, HVR-L2 and an        HVR-L3 sequence having at least 85% sequence identity to        RASQDVSTAVA (SEQ ID NO:20), SASFLYS (SEQ ID NO:21) and QQYLYHPAT        (SEQ ID NO:22), respectively.    -   (c) In a specific aspect, the sequence identity is 86%, 87%,        88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or        100%. In another aspect, the heavy chain variable region        comprises one or more framework sequences juxtaposed between the        HVRs as:        (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4),        and the light chain variable regions comprises one or more        framework sequences juxtaposed between the HVRs as:        (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4).        In yet another aspect, the framework sequences are derived from        human consensus framework sequences. In a still further aspect,        the heavy chain framework sequences are derived from a Kabat        subgroup I, II, or III sequence. In a still further aspect, the        heavy chain framework sequence is a VH subgroup III consensus        framework. In a still further aspect, one or more of the heavy        chain framework sequences is the following:

HC-FR1  (SEQ ID NO: 25) EVQLVESGGGLVQPGGSLRLSCAAS HC-FR2 (SEQ ID NO: 26) WVRQAPGKGLEWV HC-FR3  (SEQ ID NO: 27)RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR HC-FR4  (SEQ ID NO: 28) WGQGTLVTVSA.

In a still further aspect, the light chain framework sequences arederived from a Kabat kappa I, II, II or IV subgroup sequence. In a stillfurther aspect, the light chain framework sequences are VL kappa Iconsensus framework. In a still further aspect, one or more of the lightchain framework sequences is the following:

LC-FR1 (SEQ ID NO: 29) DIQMTQSPSSLSASVGDRVTITC LC-FR2 (SEQ ID NO: 30)WYQQKPGKAPKLLIY LC-FR3 (SEQ ID NO: 31) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLC-FR4 (SEQ ID NO: 32) FGQGTKVEIKR.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG1. In a still further aspect, the murine constant region isselected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region if IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect the minimal effectorfunction results from an “effector-less Fc mutation” or aglycosylation.In still a further embodiment, the effector-less Fc mutation is an N297Aor D265A/N297A substitution in the constant region.

In a still further embodiment, provided is an isolated anti-PD-L1antibody comprising a heavy chain and a light chain variable regionsequence, wherein:

-   -   (a) the heavy chain sequence has at least 85% sequence identity        to the heavy chain sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVT VSA (SEQ IDNO:23), EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVT VSSASTK (SEQID NO:40), or EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVT VSS (SEQ IDNO:41), or

-   -   (b) the light chain sequences has at least 85% sequence identity        to the light chain sequence:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO:24).

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In another aspect,the heavy chain variable region comprises one or more frameworksequences juxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yetanother aspect, the framework sequences are derived from human consensusframework sequences. In a further aspect, the heavy chain frameworksequences are derived from a Kabat subgroup I, II, or III sequence. In astill further aspect, the heavy chain framework sequence is a VHsubgroup III consensus framework. In a still further aspect, one or moreof the heavy chain framework sequences is the following:

HC-FR1  (SEQ ID NO: 25) EVQLVESGGGLVQPGGSLRLSCAAS HC-FR2 (SEQ ID NO: 26) WVRQAPGKGLEWV HC-FR3  (SEQ ID NO: 27)RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR HC-FR4  (SEQ ID NO: 28) WGQGTLVTVSA.

In a still further aspect, the light chain framework sequences arederived from a Kabat kappa I, II, II or IV subgroup sequence. In a stillfurther aspect, the light chain framework sequences are VL kappa Iconsensus framework. In a still further aspect, one or more of the lightchain framework sequences is the following:

LC-FR1 (SEQ ID NO: 29) DIQMTQSPSSLSASVGDRVTITC LC-FR2 (SEQ ID NO: 30)WYQQKPGKAPKLLIY LC-FR3 (SEQ ID NO: 31) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLC-FR4 (SEQ ID NO: 32) FGQGTKVEIKR.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG1. In a still further aspect, the murine constant region isselected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region if IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect, the minimal effectorfunction results from production in prokaryotic cells. In a stillfurther specific aspect the minimal effector function results from an“effector-less Fc mutation” or aglycosylation. In still a furtherembodiment, the effector-less Fc mutation is an N297A or D265A/N297Asubstitution in the constant region.

In a still further embodiment, the invention provides for compositionscomprising any of the above described anti-PD-L1 antibodies incombination with at least one pharmaceutically-acceptable carrier.

In a still further embodiment, provided is an isolated nucleic acidencoding a light chain or a heavy chain variable region sequence of ananti-PD-L1 antibody, wherein:

(a) the heavy chain further comprises and HVR-H1, HVR-H2 and an HVR-H3sequence having at least 85% sequence identity to GFTFSDSWIH (SEQ IDNO:17), AWISPYGGSTYYADSVKG (SEQ ID NO:18) and RHWPGGFDY (SEQ ID NO:19),respectively, and

(b) the light chain further comprises an HVR-L1, HVR-L2 and an HVR-L3sequence having at least 85% sequence identity to RASQDVSTAVA (SEQ IDNO:20), SASFLYS (SEQ ID NO:21) and QQYLYHPAT (SEQ ID NO:22),respectively.

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In aspect, theheavy chain variable region comprises one or more framework sequencesjuxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yetanother aspect, the framework sequences are derived from human consensusframework sequences. In a further aspect, the heavy chain frameworksequences are derived from a Kabat subgroup I, II, or III sequence. In astill further aspect, the heavy chain framework sequence is a VHsubgroup III consensus framework. In a still further aspect, one or moreof the heavy chain framework sequences is the following:

HC-FR1  (SEQ ID NO: 25) EVQLVESGGGLVQPGGSLRLSCAAS HC-FR2 (SEQ ID NO: 26) WVRQAPGKGLEWV HC-FR3  (SEQ ID NO: 27)RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR HC-FR4  (SEQ ID NO: 28) WGQGTLVTVSA.

In a still further aspect, the light chain framework sequences arederived from a Kabat kappa I, II, II or IV subgroup sequence. In a stillfurther aspect, the light chain framework sequences are VL kappa Iconsensus framework. In a still further aspect, one or more of the lightchain framework sequences is the following:

LC-FR1 (SEQ ID NO: 29) DIQMTQSPSSLSASVGDRVTITC LC-FR2 (SEQ ID NO: 30)WYQQKPGKAPKLLIY LC-FR3 (SEQ ID NO: 31) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLC-FR4 (SEQ ID NO: 32) FGQGTKVEIKR.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG1. In a still further aspect, the murine constant region isselected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region if IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect, the minimal effectorfunction results from production in prokaryotic cells. In a stillfurther specific aspect the minimal effector function results from an“effector-less Fc mutation” or aglycosylation. In still a furtheraspect, the effector-less Fc mutation is an N297A or D265A/N297Asubstitution in the constant region.

In another further embodiment, provided is an isolated anti-PDL1antibody comprising a heavy chain and a light chain variable regionsequence, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to theheavy chainsequence:EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWG QGTLVTVSS (SEQID NO:41), or

(b) the light chain sequences has at least 85% sequence identity to thelight chain sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASF LYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ IDNO:24).

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In another aspect,the heavy chain variable region comprises one or more frameworksequences juxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yetanother aspect, the framework sequences are derived from human consensusframework sequences. In a further aspect, the heavy chain frameworksequences are derived from a Kabat subgroup I, II, or III sequence. In astill further aspect, the heavy chain framework sequence is a VHsubgroup III consensus framework. In a still further aspect, one or moreof the heavy chain framework sequences is the following:

HC-FR1 (SEQ ID NO: 25) EVQLVESGGGLVQPGGSLRLSCAAS HC-FR2 (SEQ ID NO: 26)WVRQAPGKGLEWV HC-FR3 (SEQ ID NO: 27) RFTISADTSKNTAYLQMNSLRAEDTAVYYCARHC-FR4 (SEQ ID NO: 42) WGQGTLVTVSS.

In a still further aspect, the light chain framework sequences arederived from a Kabat kappa I, II, II or IV subgroup sequence. In a stillfurther aspect, the light chain framework sequences are VL kappa Iconsensus framework. In a still further aspect, one or more of the lightchain framework sequences is the following:

LC-FR1 (SEQ ID NO: 29) DIQMTQSPSSLSASVGDRVTITC LC-FR2 (SEQ ID NO: 30)WYQQKPGKAPKLLIY LC-FR3 (SEQ ID NO: 31) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLC-FR4 (SEQ ID NO: 32) FGQGTKVEIKR.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG1. In a still further aspect, the murine constant region isselected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region if IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect, the minimal effectorfunction results from production in prokaryotic cells. In a stillfurther specific aspect the minimal effector function results from an“effector-less Fc mutation” or aglycosylation. In still a furtherembodiment, the effector-less Fc mutation is an N297A or D265A/N297Asubstitution in the constant region.

In a further aspect, the heavy chain variable region comprises one ormore framework sequences juxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In astill further aspect, the framework sequences are derived from humanconsensus framework sequences. In a still further aspect, the heavychain framework sequences are derived from a Kabat subgroup I, II, orIII sequence. In a still further aspect, the heavy chain frameworksequence is a VH subgroup III consensus framework. In a still furtheraspect, one or more of the heavy chain framework sequences is thefollowing:

HC-FR1 (SEQ ID NO: 43) EVQLVESGGGLVQPGGSLRLSCAASGFTFS HC-FR2(SEQ ID NO: 44) WVRQAPGKGLEWVA HC-FR3 (SEQ ID NO: 27)RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR HC-FR4 (SEQ ID NO: 45) WGQGTLVTVSS.

In a still further aspect, the light chain framework sequences arederived from a Kabat kappa I, II, II or IV subgroup sequence. In a stillfurther aspect, the light chain framework sequences are VL kappa Iconsensus framework. In a still further aspect, one or more of the lightchain framework sequences is the following:

LC-FR1 (SEQ ID NO: 29) DIQMTQSPSSLSASVGDRVTITC LC-FR2 (SEQ ID NO: 30)WYQQKPGKAPKLLIY LC-FR3 (SEQ ID NO: 31) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLC-FR4 (SEQ ID NO: 46) FGQGTKVEIK.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG1. In a still further aspect, the murine constant region isselected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region if IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect the minimal effectorfunction results from an “effector-less Fc mutation” or aglycosylation.In still a further embodiment, the effector-less Fc mutation is an N297Aor D265A/N297A substitution in the constant region.

In yet another embodiment, provided is an anti-PDL1 antibody comprisinga heavy chain and a light chain variable region sequence, wherein:

-   -   (d) the heavy chain further comprises and HVR-H1, HVR-H2 and an        HVR-H3 sequence having at least 85% sequence identity to        GFTFSDSWIH (SEQ ID NO:17), AWISPYGGSTYYADSVKG (SEQ ID NO:18) and        RHWPGGFDY (SEQ ID NO:19), respectively, or    -   (e) the light chain further comprises an HVR-L1, HVR-L2 and an        HVR-L3 sequence having at least 85% sequence identity to        RASQDVSTAVA (SEQ ID NO:20), SASFLYS (SEQ ID NO:21) and QQYLYHPAT        (SEQ ID NO:22), respectively.

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In another aspect,the heavy chain variable region comprises one or more frameworksequences juxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yetanother aspect, the framework sequences are derived from human consensusframework sequences. In a still further aspect, the heavy chainframework sequences are derived from a Kabat subgroup I, II, or IIIsequence. In a still further aspect, the heavy chain framework sequenceis a VH subgroup III consensus framework. In a still further aspect, oneor more of the heavy chain framework sequences is the following:

HC-FR1 (SEQ ID NO: 25) EVQLVESGGGLVQPGGSLRLSCAAS HC-FR2 (SEQ ID NO: 26)WVRQAPGKGLEWV HC-FR3 (SEQ ID NO: 27) RFTISADTSKNTAYLQMNSLRAEDTAVYYCARHC-FR4 (SEQ ID NO: 47) WGQGTLVTVSSASTK.

In a still further aspect, the light chain framework sequences arederived from a Kabat kappa I, II, II or IV subgroup sequence. In a stillfurther aspect, the light chain framework sequences are VL kappa Iconsensus framework. In a still further aspect, one or more of the lightchain framework sequences is the following:

LC-FR1 (SEQ ID NO: 29) DIQMTQSPSSLSASVGDRVTITC LC-FR2 (SEQ ID NO: 30)WYQQKPGKAPKLLIY LC-FR3 (SEQ ID NO: 31) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLC-FR4 (SEQ ID NO: 32) FGQGTKVEIKR.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG1. In a still further aspect, the murine constant region isselected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region if IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect the minimal effectorfunction results from an “effector-less Fc mutation” or aglycosylation.In still a further embodiment, the effector-less Fc mutation is an N297Aor D265A/N297A substitution in the constant region.

In a still further embodiment, provided is an isolated anti-PDL1antibody comprising a heavy chain and a light chain variable regionsequence, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to theheavy chain sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVT VSSASTK (SEQID NO:40), or

(b) the light chain sequences has at least 85% sequence identity to thelight chain sequence:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO:24).

In some embodiments, provided is an isolated anti-PDL1 antibodycomprising a heavy chain and a light chain variable region sequence,wherein the light chain variable region sequence has at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequence identityto the amino acid sequence of SEQ ID NO:24. In some embodiments,provided is an isolated anti-PDL1 antibody comprising a heavy chain anda light chain variable region sequence, wherein the heavy chain variableregion sequence has at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity to the amino acid sequence of SEQID NO:40. In some embodiments, provided is an isolated anti-PDL1antibody comprising a heavy chain and a light chain variable regionsequence, wherein the light chain variable region sequence has at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to the amino acid sequence of SEQ ID NO:24 and the heavy chainvariable region sequence has at least 85%, at least 86%, at least 87%,at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to the amino acid sequenceof SEQ ID NO:40.

In a still further embodiment, provided is an isolated anti-PDL1antibody comprising a heavy chain and a light chain sequence, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to theheavy chain sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:48), or

(b) the light chain sequences has at least 85% sequence identity to thelight chain sequence:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:49).

In some embodiments, provided is an isolated anti-PDL1 antibodycomprising a heavy chain and a light chain sequence, wherein the lightchain sequence has at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity to the amino acid sequence of SEQID NO:49. In some embodiments, provided is an isolated anti-PDL1antibody comprising a heavy chain and a light chain sequence, whereinthe heavy chain sequence has at least 85%, at least 86%, at least 87%,at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to the amino acid sequenceof SEQ ID NO:48. In some embodiments, provided is an isolated anti-PDL1antibody comprising a heavy chain and a light chain sequence, whereinthe light chain sequence has at least 85%, at least 86%, at least 87%,at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to the amino acid sequenceof SEQ ID NO:49 and the heavy chain sequence has at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% sequence identity tothe amino acid sequence of SEQ ID NO:48.

In a still further aspect, the nucleic acid further comprises a vectorsuitable for expression of the nucleic acid encoding any of thepreviously described anti-PD-L1 antibodies. In a still further specificaspect, the vector further comprises a host cell suitable for expressionof the nucleic acid. In a still further specific aspect, the host cellis a eukaryotic cell or a prokaryotic cell. In a still further specificaspect, the eukaryotic cell is a mammalian cell, such as Chinese HamsterOvary (CHO).

The anti-PD-L1 antibody or antigen binding fragment thereof, may be madeusing methods known in the art, for example, by a process comprisingculturing a host cell containing nucleic acid encoding any of thepreviously described anti-PD-L1 antibodies or antigen-binding fragmentin a form suitable for expression, under conditions suitable to producesuch antibody or fragment, and recovering the antibody or fragment.

In a still further embodiment, the invention provides for a compositioncomprising an anti-PD-L1 antibody or antigen binding fragment thereof asprovided herein and at least one pharmaceutically acceptable carrier.

Agents that Decreases or Inhibits TIGIT Expression and/or Activity

Provided herein is a method for treatment or delaying progression ofcancer in an individual comprising administering to the individual aneffective amount of a PD-1 axis binding antagonist in combination withan agent that decreases or inhibits TIGIT expression and/or activity.Provided herein is also a method for reducing or inhibiting cancerrelapse or cancer progression in an individual comprising administeringto the individual an effective amount of a PD-1 axis binding antagonistin combination with an agent that decreases or inhibits TIGIT expressionand/or activity. Provided herein is also a method for treating ordelaying progression of an immune related disease in an individualcomprising administering to the individual an effective amount of a PD-1axis binding antagonist in combination with an agent that decreases orinhibits TIGIT expression and/or activity. Provided herein is also amethod for reducing or inhibiting progression of an immune relateddisease in an individual comprising administering to the individual aneffective amount of a PD-1 axis binding antagonist in combination withan agent that decreases or inhibits TIGIT expression and/or activity.Provided herein is also a method for increasing, enhancing orstimulating an immune response or function in an individual comprisingadministering to the individual an effective amount of a PD-1 axisbinding antagonist in combination with an agent that decreases orinhibits TIGIT expression and/or activity. Provided herein is also amethod for increasing, enhancing or stimulating an immune response orfunction in an individual comprising administering to the individual aneffective amount of an agent that decreases or inhibits TIGIT expressionand/or activity and an agent that decreases or inhibits one or moreadditional immune co-inhibitory receptors. Provided herein is also amethod for increasing, enhancing or stimulating an immune response orfunction in an individual comprising administering to the individual aneffective amount of an agent that decreases or inhibits TIGIT expressionand/or activity and an agent that increases or activates one or moreadditional immune co-stimulatory receptors. For example, agent thatdecreases or inhibits TIGIT expression and/or activity includes anantagonist of TIGIT expression and/or activity, an antagonist of PVRexpression and/or activity, an agent that inhibits and/or blocks theinteraction of TIGIT with PVR, an agent that inhibits and/or blocks theinteraction of TIGIT with PVRL2, an agent that inhibits and/or blocksthe interaction of TIGIT with PVRL3, an agent that inhibits and/orblocks the intracellular signaling mediated by TIGIT binding to PVR, anagent that inhibits and/or blocks the intracellular signaling mediatedby TIGIT binding to PVRL2, an agent that inhibits and/or blocks theintracellular signaling mediated by TIGIT binding to PVRL3, andcombinations thereof.

In some embodiments, the antagonist of TIGIT expression and/or activityincludes a small molecule inhibitor, an inhibitory antibody orantigen-binding fragment thereof, an aptamer, an inhibitory nucleicacid, and an inhibitory polypeptide.

In some embodiments, the antagonist of PVR expression and/or activityincludes a small molecule inhibitor, an inhibitory antibody orantigen-binding fragment thereof, an aptamer, an inhibitory nucleicacid, and an inhibitory polypeptide.

In some embodiments, the agent that inhibits and/or blocks theinteraction of TIGIT with PVR includes a small molecule inhibitor, aninhibitory antibody or antigen-binding fragment thereof, an aptamer, aninhibitory nucleic acid, and an inhibitory polypeptide.

In some embodiments, the agent that inhibits and/or blocks theinteraction of TIGIT with PVRL2 includes a small molecule inhibitor, aninhibitory antibody or antigen-binding fragment thereof, an aptamer, aninhibitory nucleic acid, and an inhibitory polypeptide.

In some embodiments, the agent that inhibits and/or blocks theinteraction of TIGIT with PVRL3 includes a small molecule inhibitor, aninhibitory antibody or antigen-binding fragment thereof, an aptamer, aninhibitory nucleic acid, and an inhibitory polypeptide.

In some embodiments, the agent that inhibits and/or blocks theintracellular signaling mediated by TIGIT binding to PVR includes asmall molecule inhibitor, an inhibitory antibody or antigen-bindingfragment thereof, an aptamer, an inhibitory nucleic acid, and aninhibitory polypeptide.

In some embodiments, the agent that inhibits and/or blocks theintracellular signaling mediated by TIGIT binding to PVRL2 includes asmall molecule inhibitor, an inhibitory antibody or antigen-bindingfragment thereof, an aptamer, an inhibitory nucleic acid, and aninhibitory polypeptide.

In some embodiments, the agent that inhibits and/or blocks theintracellular signaling mediated by TIGIT binding to PVRL3 includes asmall molecule inhibitor, an inhibitory antibody or antigen-bindingfragment thereof, an aptamer, an inhibitory nucleic acid, and aninhibitory polypeptide.

In some embodiments, the antagonist of TIGIT expression and/or activityis an inhibitory nucleic acid selected from an antisense polynucleotide,an interfering RNA, a catalytic RNA, and an RNA-DNA chimera.

In some embodiments, the antagonist of TIGIT expression and/or activityis an anti-TIGIT antibody or antigen-binding fragment thereof.

The anti-TIGIT antibodies useful in this invention, includingcompositions containing such antibodies, such as those described in WO2009/126688, may be used in combination with PD-1 axis bindingantagonists.

Anti-TIGIT Antibodies

The present invention provides anti-TIGIT antibodies. Exemplaryantibodies include polyclonal, monoclonal, humanized, bispecific, andheteroconjugate antibodies. It will be understood by one of ordinaryskill in the art that the invention also provides antibodies againstother polypeptides (i.e., anti-PVR antibodies) and that any of thedescription herein drawn specifically to the method of creation,production, varieties, use or other aspects of anti-TIGIT antibodieswill also be applicable to antibodies specific for other non-TIGITpolypeptides.

Polyclonal Antibodies

The anti-TIGIT antibodies may comprise polyclonal antibodies. Methods ofpreparing polyclonal antibodies are known to the skilled artisan.Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Theimmunizing agent may include the TIGIT polypeptide or a fusion proteinthereof. It may be useful to conjugate the immunizing agent to a proteinknown to be immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants which may be employed include Freund'scomplete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). The immunization protocol may beselected by one skilled in the art without undue experimentation.

Monoclonal Antibodies

The anti-TIGIT antibodies may, alternatively, be monoclonal antibodies.Monoclonal antibodies may be prepared using hybridoma methods, such asthose described by Kohler and Milstein, Nature, 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes may beimmunized in vitro.

The immunizing agent will typically include the TIGIT polypeptide or afusion protein thereof. Generally, either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell [Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103]. Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against thepolypeptide. Preferably, the binding specificity of monoclonalantibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences [U.S. Pat.No. 4,816,567; Morrison et al., supra] or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

Human and Humanized Antibodies

The anti-TIGIT antibodies of the invention may further comprisehumanized antibodies or human antibodies. Humanized forms of non-human(e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulinchains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

The antibodies may also be affinity matured using known selection and/ormutagenesis methods as described above. Preferred affinity maturedantibodies have an affinity which is five times, more preferably 10times, even more preferably 20 or 30 times greater than the startingantibody (generally murine, humanized or human) from which the maturedantibody is prepared.

Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forTIGIT, the other one is for any other antigen, and preferably for acell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537-539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared can be prepared using chemical linkage. Brennan et al., Science229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Fab′ fragments may be directly recovered from E. coli and chemicallycoupled to form bispecific antibodies. Shalaby et al., J. Exp. Med.175:217-225 (1992) describe the production of a fully humanizedbispecific antibody F(ab′)₂ molecule. Each Fab′ fragment was separatelysecreted from E. coli and subjected to directed chemical coupling invitro to form the bispecific antibody. The bispecific antibody thusformed was able to bind to cells overexpressing the ErbB2 receptor andnormal human T cells, as well as trigger the lytic activity of humancytotoxic lymphocytes against human breast tumor targets.

Various technique for making and isolating bispecific antibody fragmentsdirectly from recombinant cell culture have also been described. Forexample, bispecific antibodies have been produced using leucine zippers.Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipperpeptides from the Fos and Jun proteins were linked to the Fab′ portionsof two different antibodies by gene fusion. The antibody homodimers werereduced at the hinge region to form monomers and then re-oxidized toform the antibody heterodimers. This method can also be utilized for theproduction of antibody homodimers. The “diabody” technology described byHollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) hasprovided an alternative mechanism for making bispecific antibodyfragments. The fragments comprise a heavy-chain variable domain (V_(H))connected to a light-chain variable domain (V_(L)) by a linker which istoo short to allow pairing between the two domains on the same chain.Accordingly, the V_(H) and V_(L) domains of one fragment are forced topair with the complementary V_(L) and V_(H) domains of another fragment,thereby forming two antigen-binding sites. Another strategy for makingbispecific antibody fragments by the use of single-chain Fv (sFv) dimershas also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. As onenonlimiting example, trispecific antibodies can be prepared. See, e.g.,Tutt et al., J. Immunol. 147:60 (1991).

Exemplary bispecific antibodies may bind to two different epitopes on agiven TIGIT polypeptide herein. Alternatively, an anti-TIGIT polypeptidearm may be combined with an arm which binds to a triggering molecule ona leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32)and FcγRIII (CD16) so as to focus cellular defense mechanisms to thecell expressing the particular TIGIT polypeptide. Bispecific antibodiesmay also be used to localize cytotoxic agents to cells which express aparticular TIGIT polypeptide. These antibodies possess a TIGIT-bindingarm and an arm which binds a cytotoxic agent or a radionuclide chelator,such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody ofinterest binds the TIGIT polypeptide and further binds tissue factor(TF).

Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP03089]. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

Effector Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating cancer. For example, cysteine residue(s) may beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedmay have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195(1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimericantibodies with enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch, 53: 2560-2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design, 3: 219-230 (1989).

In some embodiment, anti-TIGIT antibodies were generated which werehamster-anti-mouse antibodies. Two antibodies, 10A7 and 1F4, alsospecifically bound to human TIGIT. The amino acid sequences of the lightand heavy chains of the 10A7 antibody were determined using standardtechniques. The light chain sequence of this antibody is:DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTK LEIKR (SEQID NO:13) and the heavy chain sequence of this antibody is:EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVT VSS (SEQ IDNO:15), where the complementarity determining regions (CDRs) of eachchain are represented by bold text. Thus, CDR1 of the 10A7 light chainhas the sequence KSSQSLYYSGVKENLLA (SEQ ID NO:1), CDR2 of the 10A7 lightchain has the sequence ASIRFT (SEQ ID NO:2), and CDR3 of the 10A7 lightchain has the sequence QQGINNPLT (SEQ ID NO:3). CDR1 of the 10A7 heavychain has the sequence GFTFSSFTMH (SEQ ID NO:4), CDR2 of the 10A7 heavychain has the sequence FIRSGSGIVFYADAVRG (SEQ ID NO:5), and CDR3 of the10A7 heavy chain has the sequence RPLGHNTFDS (SEQ ID NO:6).

The amino acid sequences of the light and heavy chains of the 1F4antibody were also determined. The light chain sequence of this antibodyis: DVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPPTFGPGTKLEVK (SEQ ID NO:14) andthe heavy chain sequence of this antibody is:EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGLIIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGLRGFYAMDYWGQGTSVT VSS (SEQ IDNO:16), where the complementarity determining regions (CDRs) of eachchain are represented by bold text. Thus, CDR1 of the 1F4 light chainhas the sequence RSSQSLVNSYGNTFLS (SEQ ID NO:7), CDR2 of the 1F4 lightchain has the sequence GISNRFS (SEQ ID NO:8), and CDR3 of the 1F4 lightchain has the sequence LQGTHQPPT (SEQ ID NO:9). CDR1 of the 1F4 heavychain has the sequence GYSFTGHLMN (SEQ ID NO:10), CDR2 of the 1F4 heavychain has the sequence LIIPYNGGTSYNQKFKG (SEQ ID NO:11), and CDR3 of the1F4 heavy chain has the sequence GLRGFYAMDY (SEQ ID NO:12).

The nucleotide sequence encoding the 1F4 light chain was determined tobe GATGTTGTGTTGACTCAAACTCCACTCTCCCTGTCTGTCAGCTTTGGAGATCAAGTTTCTATCTCTTGCAGGTCTAGTCAGAGTCTTGTAAACAGTTATGGGAACACCTTTTTGTCTTGGTACCTGCACAAGCCTGGCCAGTCTCCACAGCTCCTCATCTTTGGGATTTCCAACAGATTTTCTGGGGTGCCAGACAGGTTCAGTGGCAGTGGTTCAGGGACAGATTTCACACTCAAGATCAGCACAATAAAGCCTGAGGACTTGGGAATGTATTACTGCTTACAAGGTACGCATCAGCCTCCCACGTTCGGTCCTGGGACCAAGCTGGAGGTGAAA (SEQ ID NO:38) andthe nucleotide sequence encoding the 1F4 heavy chain was determined tobe GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAACTTCAATGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCCATCTTATGAACTGGGTGAAGCAGAGCCATGGAAAGAACCTTGAGTGGATTGGACTTATTATTCCTTACAATGGTGGTACAAGCTATAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCATCCAGCACAGCCTACATGGAGCTCCTCAGTCTGACTTCTGATGACTCTGCAGTCTATTTCTGTTCAAGAGGCCTTAGGGGCTTCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO:39).

In some embodiments, the anti-TIGIT antibody or antigen-binding fragmentthereof comprises at least one HVR comprising an amino acid sequenceselected from the amino acid sequences set forth in (1)KSSQSLYYSGVKENLLA (SEQ ID NO:1), ASIRFT (SEQ ID NO:2), QQGINNPLT (SEQ IDNO:3), GFTFSSFTMH (SEQ ID NO:4), FIRSGSGIVFYADAVRG (SEQ ID NO:5), andRPLGHNTFDS (SEQ ID NO:6), or (2) RSSQSLVNSYGNTFLS (SEQ ID NO:7), GISNRFS(SEQ ID NO:8), LQGTHQPPT (SEQ ID NO:9), GYSFTGHLMN (SEQ ID NO:10),LIIPYNGGTSYNQKFKG (SEQ ID NO:11), and GLRGFYAMDY (SEQ ID NO:12).

In some embodiments, the anti-TIGIT antibody or antigen-binding fragmentthereof, wherein the antibody light chain comprises the amino acidsequence set forth in DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTK LEIKR (SEQID NO:13) orDVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPPTFGPGTKLEVK (SEQ ID NO:14).

In some embodiments, the anti-TIGIT antibody or antigen-binding fragmentthereof, wherein the antibody heavy chain comprises the amino acidsequence set forth inEVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVT VSS (SEQ IDNO:15) or EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGLIIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGLRGFYAMDYWGQGTS VT VSS (SEQ IDNO:16).

In some embodiments, the anti-TIGIT antibody or antigen-binding fragmentthereof, wherein the antibody light chain comprises the amino acidsequence set forth in DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTK LEIKR (SEQID NO:13) orDVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPPTFGPGTKLEVK (SEQ ID NO:14) andthe antibody heavy chain comprises the amino acid sequence set forth inEVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVT VSS (SEQ IDNO:15) or EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGLIIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGLRGFYAMDYWGQGTSVT VSS (SEQ IDNO:16).

In some embodiments, the anti-TIGIT antibody or antigen-binding fragmentthereof, wherein the antibody is selected from a humanized antibody, achimeric antibody, a bispecific antibody, a heteroconjugate antibody,and an immunotoxin.

In some embodiments, the anti-TIGIT antibody or antigen-binding fragmentthereof comprises at least one HVR is at least 90% identical to an HVRset forth in any of (1) KSSQSLYYSGVKENLLA (SEQ ID NO:1), ASIRFT (SEQ IDNO:2), QQGINNPLT (SEQ ID NO:3), GFTFSSFTMH (SEQ ID NO:4),FIRSGSGIVFYADAVRG (SEQ ID NO:5), and RPLGHNTFDS (SEQ ID NO:6), or (2)RSSQSLVNSYGNTFLS (SEQ ID NO:7), GISNRFS (SEQ ID NO:8), LQGTHQPPT (SEQ IDNO:9), GYSFTGHLMN (SEQ ID NO:10), LIIPYNGGTSYNQKFKG (SEQ ID NO:11), andGLRGFYAMDY (SEQ ID NO:12).

In some embodiments, the anti-TIGIT antibody or fragment thereofcomprises the light chain and/or heavy chain comprising amino acidsequences at least 90% identical to the amino acid sequences set forthin DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTK LEIKR (SEQID NO:13) orDVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPPTFGPGTKLEVK (SEQ ID NO:14), orEVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVT VSS (SEQ IDNO:15) or EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGLIIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGLRGFYAMDYWGQGTSVT VSS (SEQ IDNO:16), respectively.

Agents that Modulate CD226 Expression and/or Activity

Provided herein is a method of treating or delaying progression ofcancer in an individual comprising administering to the individual aneffective amount of a PD-1 axis binding antagonist and an agent thatmodulates the CD226 expression and/or activity. Provided herein is alsoa method for reducing or inhibiting cancer relapse or cancer progressionin an individual comprising administering to the individual an effectiveamount of a PD-1 axis binding antagonist and an agent that modulates theCD226 expression and/or activity. Provided herein is also a method fortreating or delaying progression of an immune related disease in anindividual comprising administering to the individual an effectiveamount of a PD-1 axis binding antagonist and an agent that modulates theCD226 expression and/or activity. Provided herein is also a method forreducing or inhibiting progression of an immune related disease in anindividual comprising administering to the individual an effectiveamount of a PD-1 axis binding antagonist and agent that modulates theCD226 expression and/or activity. Provided herein is also a method ofincreasing, enhancing or stimulating an immune response or function inan individual by administering to the individual an effective amount ofa PD-1 axis binding antagonist and an agent that modulates the CD226expression and/or activity.

For example, agents that modulate the CD226 expression and/or activityare agents capable of increasing and/or stimulating CD226 expressionand/or activity, increasing and/or stimulating the interaction of CD226with PVR, PVRL2, and/or PVRL3, and increasing and/or stimulating theintracellular signaling mediated by CD226 binding to PVR, PVRL2, and/orPVRL3. In some embodiments, agents capable of increasing and/orstimulating CD226 expression and/or activity are agents that increaseand/or stimulate CD226 expression and/or activity. In some embodiments,agents capable of increasing and/or stimulating the interaction of CD226with PVR, PVRL2, and/or PVRL3 are agents that increase and/or stimulatethe interaction of CD226 with PVR, PVRL2, and/or PVRL3. In someembodiments, agents capable of increasing and/or stimulating theintracellular signaling mediated by CD226 binding to PVR, PVRL2, and/orPVRL3 are agents that increase and/or stimulate the intracellularsignaling mediated by CD226 binding to PVR, PVRL2, and/or PVRL3.

In some embodiments, the agent that modulates the CD226 expressionand/or activity is selected from an agent that inhibits and/or blocksthe interaction of CD226 with TIGIT, an antagonist of TIGIT expressionand/or activity, an antagonist of PVR expression and/or activity, anagent that inhibits and/or blocks the interaction of TIGIT with PVR, anagent that inhibits and/or blocks the interaction of TIGIT with PVRL2,an agent that inhibits and/or blocks the interaction of TIGIT withPVRL3, an agent that inhibits and/or blocks the intracellular signalingmediated by TIGIT binding to PVR, an agent that inhibits and/or blocksthe intracellular signaling mediated by TIGIT binding to PVRL2, an agentthat inhibits and/or blocks the intracellular signaling mediated byTIGIT binding to PVRL3, and combinations thereof. In some embodiments,the agent that inhibits and/or blocks the interaction of CD226 withTIGIT is selected from a small molecule inhibitor, an inhibitoryantibody or antigen-binding fragment thereof, an aptamer, an inhibitorynucleic acid, and an inhibitory polypeptide. In some embodiments, theagent that inhibits and/or blocks the interaction of CD226 with TIGIT isan anti-TIGIT antibody or antigen-binding fragment thereof. In someembodiments, the agent that inhibits and/or blocks the interaction ofCD226 with TIGIT is an inhibitory nucleic acid selected from anantisense polynucleotide, an interfering RNA, a catalytic RNA, and anRNA-DNA chimera.

In some embodiments, the antagonist of TIGIT expression and/or activityis a small molecule inhibitor, an inhibitory antibody or antigen-bindingfragment thereof, an aptamer, an inhibitory nucleic acid, and aninhibitory polypeptide. In some embodiments, the antagonist of TIGITexpression and/or activity is an anti-TIGIT antibody or antigen-bindingfragment thereof. In some embodiments, the antagonist of TIGITexpression and/or activity is an inhibitory nucleic acid selected froman antisense polynucleotide, an interfering RNA, a catalytic RNA, and anRNA-DNA chimera. In some embodiments, the antagonist of PVR expressionand/or activity is a small molecule inhibitor, an inhibitory antibody orantigen-binding fragment thereof, an aptamer, an inhibitory nucleicacid, and an inhibitory polypeptide. In some embodiments, the agent thatinhibits and/or blocks the interaction of TIGIT with PVR is a smallmolecule inhibitor, an inhibitory antibody or antigen-binding fragmentthereof, an aptamer, an inhibitory nucleic acid, and an inhibitorypolypeptide. In some embodiments, the agent that inhibits and/or blocksthe interaction of TIGIT with PVRL2 is a small molecule inhibitor, aninhibitory antibody or antigen-binding fragment thereof, an aptamer, aninhibitory nucleic acid, and an inhibitory polypeptide. In someembodiments, the agent that inhibits and/or blocks the interaction ofTIGIT with PVRL3 is a small molecule inhibitor, an inhibitory antibodyor antigen-binding fragment thereof, an aptamer, an inhibitory nucleicacid, and an inhibitory polypeptide. In some embodiments, the agent thatinhibits and/or blocks the intracellular signaling mediated by TIGITbinding to PVR is a small molecule inhibitor, an inhibitory antibody orantigen-binding fragment thereof, an aptamer, an inhibitory nucleicacid, and an inhibitory polypeptide. In some embodiments, the agent thatinhibits and/or blocks the intracellular signaling mediated by TIGITbinding to PVRL2 is a small molecule inhibitor, an inhibitory antibodyor antigen-binding fragment thereof, an aptamer, an inhibitory nucleicacid, and an inhibitory polypeptide. In some embodiments, the agent thatinhibits and/or blocks the intracellular signaling mediated by TIGITbinding to PVRL3 is a small molecule inhibitor, an inhibitory antibodyor antigen-binding fragment thereof, an aptamer, an inhibitory nucleicacid, and an inhibitory polypeptide.

In some embodiments, the antagonist of TIGIT expression and/or activityincludes a small molecule inhibitor, an inhibitory antibody orantigen-binding fragment thereof, an aptamer, an inhibitory nucleicacid, and an inhibitory polypeptide. In some embodiments, the antagonistof PVR expression and/or activity includes a small molecule inhibitor,an inhibitory antibody or antigen-binding fragment thereof, an aptamer,an inhibitory nucleic acid, and an inhibitory polypeptide. In someembodiments, the agent that inhibits the intracellular signalingmediated by TIGIT binding to PVR is selected from the group consistingof a small molecule inhibitor, an inhibitory antibody or antigen-bindingfragment thereof, an aptamer, an inhibitory nucleic acid, and aninhibitory polypeptide. In some embodiments, the antagonist of TIGITexpression and/or activity is an anti-TIGIT antibody or antigen-bindingfragment thereof. In some embodiments, the antagonist of TIGITexpression and/or activity is an inhibitory nucleic acid selected froman antisense polynucleotide, an interfering RNA, a catalytic RNA, and anRNA-DNA chimera.

Combinations of T Cell Targets for Immunoregulatory Antibody Therapy

In addition to specific antigen recognition through the TCR, T-cellactivation is regulated through a balance of positive and negativesignals provided by co-stimulatory receptors. These surface proteins aretypically members of either the TNF receptor or B7 superfamilies.Activating co-stimulatory receptors include CD226, CD28, OX40, GITR,CD137, CD27, HVEM, MICA, ICOS, NKG2D, and 2B4. Inhibitory co-stimulatoryreceptors include CTLA-4, PD-1, TIM-3, BTLA, VISTA, LAG-3, B7H4, andCD96. Agonistic antibodies directed against activating co-stimulatorymolecules and blocking antibodies against negative co-stimulatorymolecules may enhance T-cell stimulation to promote tumor destruction.

Provided herein is a method of increasing, enhancing or stimulating animmune response or function in an individual by administering to theindividual an effective amount of an agent that decreases or inhibitsTIGIT expression and/or activity and an agent that decreases or inhibitsone or more additional immune co-inhibitory receptors. In someembodiments, the one or more additional immune co-inhibitory receptor isselected from PD-1, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, and CD96. Insome embodiments, the one or more additional immune co-inhibitoryreceptor is selected from PD-1, CTLA-4, LAG3 and TIM3.

Provided herein is also a method of increasing, enhancing or stimulatingan immune response or function in an individual by administering to theindividual an effective amount of an agent that decreases or inhibitsTIGIT expression and/or activity and an agent that increases oractivates one or more additional immune co-stimulatory receptor. In someembodiments, the one or more additional immune co-stimulatory receptoris selected from CD226, OX-40, CD28, CD27, CD137, HVEM, GITR, MICA,ICOS, NKG2D, and 2B4. In some embodiments, the one or more additionalimmune co-stimulatory receptor is selected from CD226, OX-40, CD27,CD137, HVEM and GITR. In some embodiments, the one or more additionalimmune co-stimulatory receptor is selected from OX-40 and CD27.

IV Kits

In another aspect, provided is a kit comprising a PD-1 axis bindingantagonist and a package insert comprising instructions for using thePD-1 axis binding antagonist in combination with an agent that decreasesor inhibits TIGIT expression and/or activity to treat or delayprogression of cancer in an individual or for enhancing immune functionof an individual having cancer. Any of the PD-1 axis binding antagonistsand/or agents that decreases or inhibits TIGIT expression and/oractivity described herein may be included in the kit.

In another aspect, provided is a kit comprising a PD-1 axis bindingantagonist and an agent that decreases or inhibits TIGIT expressionand/or activity, and a package insert comprising instructions for usingthe PD-1 axis binding antagonist and the agent that decreases orinhibits TIGIT expression and/or activity to treat or delay progressionof cancer in an individual or for enhancing immune function of anindividual having cancer. Any of the PD-1 axis binding antagonistsand/or agents that decreases or inhibits TIGIT expression and/oractivity described herein may be included in the kit.

In another aspect, provided is a kit comprising an agent that decreasesor inhibits TIGIT expression and/or activity and a package insertcomprising instructions for using the agent that decreases or inhibitsTIGIT expression and/or activity in combination with a PD-1 axis bindingantagonist to treat or delay progression of cancer in an individual orfor enhancing immune function of an individual having cancer. Any of thePD-1 axis binding antagonists and/or agents that decreases or inhibitsTIGIT expression and/or activity described herein may be included in thekit.

In another aspect, provided is a kit comprising a PD-1 axis bindingantagonist and a package insert comprising instructions for using thePD-1 axis binding antagonist in combination with an agent that modulatesthe CD226 expression and/or activity to treat or delay progression ofcancer in an individual. Any of the PD-1 axis binding antagonists and/oragents that modulate the CD226 expression and/or activity describedherein may be included in the kit.

In another aspect, provided is a kit comprising a PD-1 axis bindingantagonist and an agent that modulates the CD226 expression and/oractivity, and a package insert comprising instructions for using thePD-1 axis binding antagonist and the agent that modulates the CD226expression and/or activity to treat or delay progression of cancer in anindividual. Any of the PD-1 axis binding antagonists and/or agents thatmodulate the CD226 expression and/or activity described herein may beincluded in the kit.

In another aspect, provided is a kit comprising an agent that modulatesthe CD226 expression and/or activity and a package insert comprisinginstructions for using the agent modulates the CD226 expression and/oractivity in combination with a PD-1 axis binding antagonist to treat ordelay progression of cancer in an individual. Any of the PD-1 axisbinding antagonists and/or agents that modulate the CD226 expressionand/or activity described herein may be included in the kit.

In another aspect, provided is a kit comprising a PD-1 axis bindingantagonist and a package insert comprising instructions for using thePD-1 axis binding antagonist in combination with an agent that modulatesthe CD226 expression and/or activity to enhance immune function of anindividual having cancer. Any of the PD-1 axis binding antagonistsand/or agents that modulate the CD226 expression and/or activitydescribed herein may be included in the kit.

In another aspect, provided is a kit comprising a PD-1 axis bindingantagonist and an agent that modulates the CD226 expression and/oractivity, and a package insert comprising instructions for using thePD-1 axis binding antagonist and the agent that modulates the CD226expression and/or activity to enhance immune function of an individualhaving cancer. Any of the PD-1 axis binding antagonists and/or agentsthat modulate the CD226 expression and/or activity described herein maybe included in the kit.

In another aspect, provided is a kit comprising an agent modulates theCD226 expression and/or activity and a package insert comprisinginstructions for using the agent that modulates the CD226 expressionand/or activity in combination with a PD-1 axis binding antagonist toenhance immune function of an individual having cancer. Any of the PD-1axis binding antagonists and/or agents that modulate the CD226expression and/or activity described herein may be included in the kit.

In another aspect, provided is a kit comprising an agent that decreasesor inhibits TIGIT expression and/or activity and a package insertcomprising instructions for using the agent that decreases or inhibitsTIGIT expression and/or activity in combination with an agent thatdecreases or inhibits one or more additional immune co-inhibitoryreceptors to treat or delay progression of cancer in an individual or toenhance immune function of an individual having cancer. Any of theagents that decrease or inhibit TIGIT expression and/or activity and/oragents that decrease or inhibit one or more additional immuneco-inhibitory receptors described herein may be included in the kit.

In another aspect, provided is a kit comprising an agent that decreasesor inhibits TIGIT expression and/or activity and an agent that decreasesor inhibits one or more additional immune co-inhibitory receptors, and apackage insert comprising instructions for using the agent thatdecreases or inhibits TIGIT expression and/or activity and the agentthat decreases or inhibits one or more additional immune co-inhibitoryreceptors to treat or delay progression of cancer in an individual or toenhance immune function of an individual having cancer. Any of theagents that decrease or inhibit TIGIT expression and/or activity and/oragents that decrease or inhibit one or more additional immuneco-inhibitory receptors described herein may be included in the kit.

In another aspect, provided is a kit comprising an agent that decreasesor inhibits one or more additional immune co-inhibitory receptors and apackage insert comprising instructions for using the agent thatdecreases or inhibits one or more additional immune co-inhibitoryreceptors in combination with an agent that decreases or inhibits TIGITexpression and/or activity to treat or delay progression of cancer in anindividual or to enhance immune function of an individual having cancer.Any of the agents that decrease or inhibit TIGIT expression and/oractivity and/or agents that decrease or inhibit one or more additionalimmune co-inhibitory receptors described herein may be included in thekit.

In another aspect, provided is a kit comprising an agent that decreasesor inhibits TIGIT expression and/or activity and a package insertcomprising instructions for using the agent that decreases or inhibitsTIGIT expression and/or activity in combination with an agent thatincreases or activates one or more additional immune co-stimulatoryreceptors to treat or delay progression of cancer in an individual or toenhance immune function of an individual having cancer. Any of theagents that decrease or inhibit TIGIT expression and/or activity and/oragents that increase or activate one or more additional immuneco-stimulatory receptors described herein may be included in the kit.

In another aspect, provided is a kit comprising an agent that decreasesor inhibits TIGIT expression and/or activity and an agent that increasesor activates one or more additional immune co-stimulatory receptors, anda package insert comprising instructions for using the agent thatdecreases or inhibits TIGIT expression and/or activity and the agentthat increases or activates one or more additional immune co-stimulatoryreceptors to treat or delay progression of cancer in an individual or toenhance immune function of an individual having cancer. Any of theagents that decrease or inhibit TIGIT expression and/or activity and/oragents that increase or activate one or more additional immuneco-stimulatory receptors described herein may be included in the kit.

In another aspect, provided is a kit comprising an agent that increasesor activates one or more additional immune co-stimulatory receptors anda package insert comprising instructions for using the agent thatincreases or activates one or more additional immune co-stimulatoryreceptors in combination with an agent that decreases or inhibits TIGITexpression and/or activity to treat or delay progression of cancer in anindividual or to enhance immune function of an individual having cancer.Any of the agents that decrease or inhibit TIGIT expression and/oractivity and/or agents that increase or activate one or more additionalimmune co-stimulatory receptors described herein may be included in thekit.

In some embodiments, the kit comprises a container containing one ormore of the PD-1 axis binding antagonists and agents that decreases orinhibits TIGIT expression and/or activity described herein. In someembodiments, the kit comprises a container containing one or more of thePD-1 axis binding antagonists and agents that modulates CD226 expressionand/or activity described herein. In some embodiments, the kit comprisesa container containing one or more of the agents that decrease orinhibit TIGIT expression and/or activity and agents that decrease orinhibit one or more additional immune co-inhibitory receptors describedherein. In some embodiments, the kit comprises a container containingone or more of the agents that decrease or inhibit TIGIT expressionand/or activity and agents that increase or activate one or moreadditional immune co-stimulatory receptors described herein. Suitablecontainers include, for example, bottles, vials (e.g., dual chambervials), syringes (such as single or dual chamber syringes) and testtubes. The container may be formed from a variety of materials such asglass or plastic. In some embodiments, the kit may comprise a label(e.g., on or associated with the container) or a package insert. Thelabel or the package insert may indicate that the compound containedtherein may be useful or intended for treating or delaying progressionof cancer in an individual or for enhancing immune function of anindividual having cancer. The kit may further comprise other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, and syringes.

EXAMPLES

The invention can be further understood by reference to the followingexamples, which are provided by way of illustration and are not meant tobe limiting.

Example 1 TIGIT is Highly Expressed on Exhausted CD8⁺ and CD4+ T Cellsand Correlated with PD-1 Expression

To confirm that CD8⁺ T cells are competent to express TIGIT afterstimulation in vitro, MACS-enriched C57BL6/J splenic CD8⁺ T cells werestimulated with plate-bound anti-CD3 and anti-CD28 for 24-48 hours invitro. Flow cytometry was used to measure TIGIT expression. In line withTIGIT's expression by CD4⁺ T cells (Yu, X., et al. The surface proteinTIGIT suppresses T cell activation by promoting the generation of matureimmunoregulatory dendritic cells. Nature immunology 10, 48-57 (2009)),murine CD8⁺ T cells expressed TIGIT within 48 hours of stimulation invitro (FIG. 1A-1D).

To assess TIGIT expression by activated CD8⁺ T cells in vivo, C57BL6/Jmice were infected with Armstrong strain Lymphocytic ChoriomeningitisVirus (LCMV), and splenocytes were analyzed 7 days after infection.Briefly, for acute infections, mice were intravenously infected with2×10⁶ plaque-forming units (PFU) Armstrong strain LCMV. Flow cytometrywas used to measure TIGIT expression by naïve (CD44^(low) CD62L^(high))and effector memory (CD44^(high) CD62L^(low)) CD8⁺ and CD4+ T cells. Atthe peak of the LCMV T cell response, a subset of CD4⁺ effector memory Tcells (T_(EM)) and nearly all CD8⁺ T_(EM) cells strongly expressed TIGIT(FIG. 1E-1F). Flow cytometry was used to measure TIGIT expression byPD-1^(high) and PD-1^(low) effector memory CD8⁺ T cells. Interestingly,TIGIT expression was near perfectly correlated with PD-1 expression(FIG. 1G-1H).

Because PD-1 is associated with T cell exhaustion, TIGIT expression wasexamined on chronically stimulated T cells. Briefly, for chronicinfections, C57BL6/J mice were intravenously infected with 2×10⁶ PFUClone 13 strain LCMV and treated with 500 ug and 250 ug of depletinganti-CD4 antibodies (clone GK1.5) 3 days before and 4 days afterinfection, respectively. Where indicated, mice infected with Clone 13strain LCMV received intraperitoneal injections of 200 ug of isotypecontrol antibodies, 200 ug of anti-PD-L1 antibodies, and/or 500 ug ofanti-TIGIT antibodies 3 times per week from days 28 to 42post-infection. Splenocytes were analyzed 42 days after infection. Flowcytometry was used to measure TIGIT expression by naïve (CD44^(low)CD62L^(high)), central memory (CD44^(high) CD62L^(high)) and effectormemory (CD44^(high) CD62L^(low)) CD8⁺ T cells. Indeed, in micechronically infected with Clone 13 strain LCMV, TIGIT was highlyexpressed predominantly on PD-1^(high) T cells but not on naïve cells,PD-1^(low) T_(EM) cells, or central memory T cells (FIG. 1I-1J).

Example 2 A Role of TIGIT in T Cell Exhaustion in TIGIT Deficient Mice

To characterize the role of TIGIT in T cell exhaustion, mice in whichTIGIT was conditionally deleted in T cells were generated (TIGIT^(fl/fl)CD4-cre⁺ (CKO), FIG. 2). Briefly, CD4^(cre) mice and TIGIT^(loxP/loxP)mice were generated on a C57BL/6J background with standard techniquesand crossed. The quality-tested ES cell line (Art B6/3.6 (geneticbackground: C57BL/6 NTac) was grown on a mitotically inactivated feederlayer comprised of mouse embryonic fibroblasts in ES cell culture mediumcontaining Leukemia inhibitory factor and Fetal Bovine Serum. The cellswere electroporated with the linearized DNA targeting vector accordingto Taconic Artemis' Standard Operation Procedures. G418 and Gancyclovirselection were used as mechanisms for enrichment of homologouslyrecombined clones. Resistant ES cell colonies (ES clones) with adistinct morphology were isolated on day 8 after transfection andanalysed by Southern Blotting and/or PCR in a primary screen. Homologousrecombinant ES cell clones were expanded and frozen in liquid nitrogenafter extensive molecular validation. The neo cassette was removed byflpE recombinase before microinjection into Bl/6 female albino donors.Chimeric offspring were produced and tails were screened by PCR forgermline transmission. TIGIT expression was ablated with 96% efficiencyfrom T cells in TIGIT^(loxP/loxP) CD4^(cre) mice.

Mice whose T cells lacked TIGIT mounted a CD4⁺ and CD8⁺ T cell responseto acute Armstrong strain LCMV infection that was similar to wild-typemice (FIG. 3A-3L).

To assess the effect in a chronic infection setting, TIGIT^(fl/fl)CD4-cre⁻ (WT) and TIGIT^(fl/fl) CD4-cre⁺ (CKO) mice were brieflydepleted of CD4⁺ T cells and infected with Clone 13 strain LCMV.Splenocytes and liver viral titers were analyzed 42 days afterinfection. After chronic infection with Clone 13 strain LCMV,significantly more CD8⁺ and CD4⁺ T cells from TIGIT^(fl/fl) CD4-cre⁺(CKO) mice were competent to produce interferon gamma (IFN γ) than wereT cells from wildtype littermate mice (TIGIT^(fl/fl) CD4-cre⁻ (WT))(82-86% increase, P<0.01, FIG. 4A-4L). Furthermore, viral loads weresignificantly reduced in chronically infected TIGIT^(fl/fl) CD4-cre⁺(CKO) mice (68% decrease, P<0.0001, FIG. 4M).

These results suggest that TIGIT plays an important role in regulating Tcell activity and response during chronic immune responses such asduring a chronic viral infection, and that TIGIT can regulate theeffector function, in particular the competency to produce effectorcytokines, such as IFNγ and TNFα, of chronically stimulated or exhaustedCD8⁺ and CD4⁺ T cells.

Example 3 TIGIT and PD-1 Synergistically Regulate the Effector Functionof Exhausted T Cells In Vivo

Since TIGIT expression was closely correlated with PD-1 expression,especially in CD8⁺ T cells during acute and chronic viral infection(FIG. 1A-1J), blocking TIGIT and PD-1 in combination may restore T celleffector function to greater levels than would be obtained by blockingeither co-receptor singly.

To test this hypothesis, C57BL6/J mice were briefly depleted of CD4⁺ Tcells and infected with Clone 13 strain LCMV. For chronic infections,mice were intravenously infected with 2×10⁶ PFU Clone 13 strain LCMV andtreated with 500 ug and 250 ug of depleting anti-CD4 antibodies (cloneGK1.5) 3 days before and 4 days after infection, respectively. Whereindicated, mice infected with Clone 13 strain LCMV receivedintraperitoneal injections of 200 ug of isotype control antibodies, 200ug of anti-PD-L1 antibodies, and/or 500 ug of anti-TIGIT antibodies 3times per week from days 28 to 42 post-infection. Treatment was startedat 28 days post-infection because the T cell response is largelyexhausted at this time-point in this model of chronic viral infection(Wherry et al, Molecular Signature of CD8+ T cell Exhaustion DuringChronic Viral Infection, Immunity. 2007 October; 27(4):670-84).Splenocytes and liver viral titers were analyzed 42 days afterinfection.

In these mice, anti-PD-L1 treatment induced more robust CD8⁺ T cellactivation than did treatment with matched isotype control antibodies(88% increase, P<0.0001, FIG. 4N-4R), as previously reported (Barber, D.L., et al. Restoring function in exhausted CD8 T cells during chronicviral infection. Nature 439, 682-687 (2006)). Anti-TIGIT treatment hadno apparent effect on CD8⁺ T cell activation on its own or incombination with anti-PD-L1 (FIG. 4N-4R). Similarly, blockade of PD-1alone moderately increased CD8⁺ T cell cytokine competency, whereasblockade of TIGIT alone had no effect (FIG. 4S-4W). However, thefrequency of IFNγ-producing CD8⁺ T cells was increased dramatically inmice treated with both anti-TIGIT and anti-PD-L1, and to a significantlygreater extent than seen in mice treated with anti-PD-L1 alone (FIG.4S-4W 93% increase, P=0.0050). A similar effect was observed with CD4⁺ Tcells (FIG. 5A-5J). As also shown in FIG. 31A-31G, TIGIT/PD-L1co-blockade significantly enhanced CD8⁺ T cell effector function, butnot CD4⁺ T cell effector function, in mice compared to mice treated withanti-PD-L1 alone. Similar effects were also observed on T cell expansionand effector function in LCMV gp33 antigen-specific T cells (FIG.31A-31G). These results demonstrate a strong synergy between PD-1 andTIGIT on exhausted CD8⁺ T cells, and indicate that TIGIT specificallyregulates CD8⁺ T cell cytokine competency and effector function.

Consistent with these results, LCMV viral loads were moderately reducedin mice treated with anti-PD-L1 alone, not reduced in mice treated withanti-TIGIT alone, and substantially reduced in mice treated with bothanti-TIGIT and anti-PD-L1 (68% viral titer reduction with anti-PD-L1treatment, P=0.0004. 92% viral titer reduction with anti-TIGIT+anti-PD-L1 treatment, P<0.0001, FIG. 4X). These data demonstrate astrong synergy between the inhibitory effects of PD-1 and TIGIT, andsuggest that unlike PD-1, TIGIT is not a broad inhibitor of effector Tcell activation, but rather has a restricted role in limiting T cellcytokine competency and effector function.

Example 4 TIGIT Expression is Elevated in Human Breast Cancer andCorrelated with Expression of CD8 and Inhibitory Co-Receptors

T cell exhaustion is also a major immunological feature of cancer, withmany tumor-infiltrating lymphocytes (TILs) expressing high levels ofinhibitory co-receptors and lacking the capacity to produce effectorcytokines (Wherry, E. J. T cell exhaustion. Nature immunology 12,492-499 (2011); Rabinovich, G. A., Gabrilovich, D. & Sotomayor, E. M.Immunosuppressive strategies that are mediated by tumor cells. Annualreview of immunology 25, 267-296 (2007)).

To determine if TIGIT inhibits TIL effector function, breast cancer geneexpression microarray data generated by the Cancer Genome Atlas Network(CGAN) was analyzed (Network, C. G. A. Comprehensive molecular portraitsof human breast tumours. Nature 490, 61-70 (2012)).

TIGIT expression was significantly elevated in breast tumors overall(135% increase relative to normal samples, P=6×10⁻¹², FIG. 6A-6B) andacross the four major molecular subtypes of breast cancer (FIG. 6A-6B)(Perou, C. M., et al. Molecular portraits of human breast tumours.Nature 406, 747-752 (2000); Sorlie, T., et al. Gene expression patternsof breast carcinomas distinguish tumor subclasses with clinicalimplications. Proceedings of the National Academy of Sciences of theUnited States of America 98, 10869-10874 (2001)). Expression of TIGITwas highly correlated with expression of CD3E, consistent with itsexpression by TILs (R²=0.61, FIG. 6C). Interestingly, TIGIT expressionwas highly correlated with CD8α but not with CD4, or only moderatelycorrelated with CD4, suggesting that TIGIT might primarily regulate CD8⁺TIL function (CD8α, R²=0.80. CD4, R²=0.42. FIG. 6D-6E).

Given the co-expression of TIGIT and PD-1 during chronic viralinfection, we also assessed the correlation of PD-1 and other inhibitoryco-receptors with TIGIT in breast cancer. Correlation between TIGIT andPD-1, CTLA4, and LAG3 was very strong (PD-1, R²=0.87. CTLA4, R²=0.76.LAG3, R²=0.80. FIG. 6F-6H). Collectively, these data suggested thatTIGIT was expressed by TILs, especially CD8⁺ T cells, and that it mightsuppress their function.

Example 5 TIGIT and PD-1 Inhibit Anti-Tumor T Cell Responses

To better characterize TIGIT by TILs in mice, BALB/C mice wereinoculated with CT26 colorectal carcinoma cells. Briefly, BALB/c micewere subcutaneously inoculated with 1×10⁵ CT26 colon carcinoma cellssuspended in matrigel (BD Biosciences) into the right unilateralthoracic flank. After two weeks, mice bearing tumors of approximately200 mm³ were randomly recruited into treatment groups receiving 35 mg/kgof isotype control antibodies, anti-PD-L1 antibodies, and/or anti-TIGITantibodies by intraperitoneal injection 3 times per week for 3 weeks.Tumors were measured 2 times per week by caliper. Animals whose tumorsbecame ulcerated/necrotic or grew larger than 2000 mm³ were euthanized.Splenocytes and tumor-infiltrating lymphocytes (TILs) were analyzed 14days after inoculation, when tumors had reached approximately 200 mm³ insize.

Consistent with TIGIT expression in human tumors (FIG. 6A-6H), both CD8⁺and CD4⁺ CT26 TILs expressed high levels of TIGIT (FIG. 7A-7D).Furthermore, in line with the chronic viral infection studies, TIL TIGITexpression was tightly correlated with expression of other inhibitoryco-receptors including PD-1 (FIG. 7A-7D) and Tim-3 (FIG. 8A-8D). Asimilar pattern of TIGIT expression was found in MC38 colon carcinomatumors (FIG. 9A-9D).

To test the physiological relevance of TIGIT expression in the contextof an anti-tumor immune response, BALB/C mice with established CT26tumors (approximately 200 mm³ in size) were treated with 200 ug isotypecontrol, 200 ug anti-PD-L1, 500 ug anti-TIGIT, or 200 ug anti-PD-L1+500ug anti-TIGIT antibodies for three weeks.

CT26 tumor growth was only slightly slowed by treatment with anti-TIGITor anti-PD-L1 alone, both of which resulted in a modest 3 day increasein median survival (FIG. 7E-7F). However, combination therapy with bothanti-PD-L1 and anti-TIGIT dramatically reduced tumor growth (75%decrease in median tumor volume by day 16, P<0.0001, FIG. 7E and FIG.10). Moreover, 70% of the mice receiving both anti-TIGIT and anti-PD-L1experienced complete and durable tumor remission and survived for theduration of the study, even in the absence of further antibody treatment(FIG. 7E-7F). These effects were also observed in tumor-bearing micetreated with a combination of blocking antibodies against TIGIT andPD-1.

To test the immunity of these surviving mice to CT26 tumor cells,approximately 60 days after initial inoculation, mice in completeremission (CR) that had received anti-TIGIT+ anti-PD-L1, as well asnaïve BALB/c mice, were re-inoculated with CT26 cells in their left (notpreviously inoculated) unilateral thoracic flanks. These mice were alsoinoculated with 1×10⁵ EMT6 breast carcinoma cells in matrigel into thefourth mammary fat pad. Tumors were measured 2 times per week. Animalswhose tumors became ulcerated/necrotic or whose total tumor burdenexceeded 2000 m³ were euthanized.

As shown in FIG. 7G-7K, both tumors grew readily in naïve control mice,but only EMT6 tumors grew in mice that had previously cleared a CT26tumor. These results indicated that co-blockade of TIGIT and PD-1 duringtumorigenesis established a state of specific immunity to CT26 tumorcells.

To determine if the efficacy of TIGIT/PD-L1 co-blockade was mediated byCD8⁺ T cells, CT26-tumor bearing mice were subjected to CD8⁺ T cellablation using depleting antibodies at the initiation of treatment withanti-TIGIT and anti-PDL1. Mice treated with anti-TIGIT and anti-PD-L1antibodies were unable to reject CT26 tumors when depleted of CD8⁺ Tcells at the start of treatment (1532% increase in mean tumor volumeafter 17 days of treatment, P=0.0004, FIG. 32A-32E). Additionally, CD8⁺T cell depletion impaired the ability of previously treated CR mice tocontrol re-inoculated CT26 tumors (FIG. 32F-32I). Taken together, theseresults demonstrated that anti-TIGIT and anti-PD-L1 acted through CD8⁺ Tcells to elicit effective primary and secondary anti-tumor immuneresponses.

To determine if PVR expression of tumor cells is dispensable forTIGIT/PD-L1 co-blockade efficacy, wildtype BALB/c mice were inoculatedwith wildtype CT26 tumors (which express PVR) or PVR-deficient CT26tumors. Briefly, wildtype CT26 tumor cells were transiently transfectedwith a nucleic acid that reduced expression of PVR. Approximately twoweeks after transfection, CT26 cells were subcloned on the basis of lossof PVR expression by flow cytometry and qPCR. When tumors reached150-200 mm³ in size, mice were treated with anti-TIGIT and anti-PD-L1antibodies, or isotype-matched control antibodies. Mice treated withanti-TIGIT and anti-PD-L1 antibodies were able to reject both wildtypeand PVR-deficient tumors, as compared to tumor-inoculated mice treatedwith control antibodies (FIG. 33). These results demonstrated thatanti-TIGIT and anti-PD-L1 act independently of tumor-expressed PVR.

The efficacy of TIGIT/PD-L1 co-blockade in the MC38 tumor model was alsotested and confirmed. Wildtype C57BL6/J mice were subcutaneouslyinoculated with syngeneic MC38 colorectal carcinoma cells and treatedestablished tumors with a combination of TIGIT and PD-L1 blockingantibodies, as before. Unlike the CT26 model, treatment with anti-PD-L1alone was sufficient to induce a complete response in some mice (FIG.25A-25G). However, as in the CT26 model, treatment of MC38 tumor-bearingmice with both anti-TIGIT and anti-PD-L1 synergistically reversed tumorgrowth and induced tumor clearance in most mice (FIG. 26A-26L). Theseeffects were also observed in mice inoculated with syngeneic EMT6 breastcarcinoma cells (FIG. 34).

These results demonstrated that co-blockade of TIGIT and PD-1 couldelicit a sustained and antigen-specific anti-tumor immune response.These results also suggested that adaptive anti-tumor responses werefully functionally and reactivated in therapeutically treated mice.

To assess the functional effects of TIGIT and PD-1 blockades on thetumor-infiltrating lymphocytes themselves, mice were inoculated withCT26 tumor cells and treated with anti-TIGIT and/or anti-PD-L1 asbefore. Seven days after the start of treatment, tumors andtumor-draining lymph nodes were collected for analysis by flowcytometry.

Tumor-infiltrating and tumor-draining lymph node resident CD4⁺ T cellsproduced little IFNγ, and did not produce more upon TIGIT/PD-1 blockade(FIG. 11A-11F). However, tumor-infiltrating CD8⁺ T cells from micetreated with both anti-TIGIT and anti-PD-L1, but not those from micetreated with anti-TIGIT or anti-PD-L1 alone, were significantly morecompetent to produce IFNγ upon stimulation in vitro (174% increaserelative to control, P=0.0001, FIG. 13H-13I). Similar results wereobserved for CD8⁺ TIL production of TNFα (FIG. 12A-12C).

Interestingly, mice treated with either anti-TIGIT or anti-PD-L1 alone,or both, all saw increased cytokine competency of tumor-draining lymphnode resident CD8⁺ T cells (75-113% increase, P<0.001, FIG. 13A-13I),suggesting that lymph node-resident CD8+ T cells were under lesserdegree of suppression than their tumor-infiltrating counterparts.Accumulation and phenotypic activation of tumor-infiltrating andtumor-draining lymph node resident CD8+ T cells and CD4+ T cells wereunchanged and weakly enhanced by single antibody treatment and dualantibody and dual antibody treatment, respectively (FIG. 12A-13I). Thefrequencies of IFNγ/TNFα dual-producing CD8⁺ T cells in tumors andtumor-draining lymph nodes followed similar patterns (FIG. 35A-35D).

Consequently, while blockade of either TIGIT or PD-L1 alone wassufficient to enhance CD8⁺ T cell effector function in tumor-draininglymph nodes, blockade of both receptors was necessary to restore thefunction of exhausted CD8⁺ T cells within the tumor itself, consistentwith the notion that tumor microenvironments are highlyimmunosuppressive.

Example 6 TIGIT Co-Expression with CD226 on Tumor-Infiltrating CD8+ TCells

TIGIT competes with the co-stimulatory receptor CD226 for binding toPoliovirus Receptor (PVR) (Yu, X., et al. The surface protein TIGITsuppresses T cell activation by promoting the generation of matureimmunoregulatory dendritic cells. Nature immunology 10, 48-57 (2009).Given that CD226 deficiency can enhance T cell exhaustion during chronicviral infection (Cella, M., et al. Loss of DNAM-1 contributes to CD8+T-cell exhaustion in chronic HIV-1 infection. European Journal ofImmunology 40(4), 949-954 (2010); Welch, M., et al. CD8 T cell defect ofTNA-a and IL-2 in DNAM-1 deficient mice delays clearance in vivo of apersistent virus infection. Virology 429(2) 163-170 (2012)), it ispossible that TIGIT may inhibit T cell responses in part by interferingwith CD226 activity.

To evaluate whether there is a relationship between CD226 and TIGIT ininhibiting T cell responses, the expression of TIGIT and CD226 wasdetermined on tumor infiltrating CD8+T cells.

As shown in FIG. 14, C57BL6/J mice were inoculated with MC38 colorectalcarcinoma cells. Splenocytes and tumor-infiltrating lymphocytes (TILs)were analyzed by FACs analysis approximately 14 days after inoculation,when tumors had reached approximately 200 mm3 in size. Representativehistogram of CD226 expression by splenic B cells (gray), splenic CD8+ Tcells (blue), and TIGIT+ tumor-infiltrating CD8+ T cells (red). Data arerepresentative of two independent experiments; n=5. FIG. 14 illustratesthat splenic CD8+ T cells highly express CD226 and furthermore, thattumor-infiltrating TIGIT+ CD8+ T cells also highly expressed CD226. Thedata demonstrates that TIGIT and CD226 are coordinately expressed onmurine tumor-infiltrating CD8⁺ T cells, and may regulate each other'sfunction on CD8⁺ T cells. This observation is similar to that inactivated CD4⁺ T cells and NK cells, which also co-express TIGIT andCD226.

Example 7 Co-Immunoprecipitation of TIGIT and CD226 on Transfected Cells

To determine whether TIGIT interacts with CD226 at the cell surface,cells were co-transfected with human-TIGIT and human-CD226 and subjectedto immunoprecipitation. Briefly, COS 7 Cells in 15 cm plates wereco-transfected with expression plasmids containing the cDNA for eitherTIGIT-HA (5 ng) or CD226-Flag (10 ng) tagged proteins, or a controlplasmid (pRK). 23 hrs after transfection the cells were washed with PBSand harvested in 4 ml of ice cold PBS and centrifuged at 300×g for 5 minand cell pellets were re-suspended in 2 ml of Lysis buffer at 4° C. Thecells were lysed over 50 min with vortexing every 15 min andsubsequently centrifuged at 10,00×g for 15 min at 4° C. The resultantsupernatant was pre-cleared with 160 μl of CL6B sepahrose slurry byrotating for 30 min at 4° C., and centrifuged for 2 min at 3000×g. Thesupernatant was equally split into two tubes and immuno-precipitatedwith either an anti-HA or an anti-flag using standard procedures. Theimmune-precipitated proteins were subjected to SDS-PAGE and westernblotted. Western blots were probed with either anti-Flag-HRP oranti-HA-HRP.

As shown in FIG. 15, anti-TIGIT pulled down CD226 and anti-CD226 pulleddown TIGIT, demonstrating that TIGIT and CD226 are in physical contactat the cell surface.

Example 8 TIGIT and CD226 Interact in Primary CD8+ T Cells

In addition to demonstrating the ability of CD226 and TIGIT to interactin transfected cells, the interaction of CD226 and TIGIT in primary CD8+T cells was also evaluated. Briefly, MACS-enriched splenic C57BL6/J CD8+T cells were stimulated with plate-bound anti-CD3 and anti-CD28antibodies and recombinant IL-2 for 48 hours and lysed. Cell lysateswere immunoprecipitated with anti-TIGIT and probed with anti-CD226. FIG.16 illustrates that TIGIT and CD226 interact in activated primary CD8+cells as both were detectable in the co-immunoprecipitate. This datademonstrates that CD226 and TIGIT also interact with each other onprimary cells.

Example 9 TIGIT/CD226 Interaction on Transfected Cells Using TR-FRET(Time Resolved-Fluorescence Resonance Energy Transfer)

To assess whether there was any molecular interaction between TIGIT andCD226, TR-FRET methodology was employed. FRET (Fluorescence ResonanceEnergy Transfer) is based on the transfer of energy between twofluorophores, a donor and an acceptor, when in close proximity.Molecular interactions between biomolecules can be assessed by couplingeach partner with a fluorescent label and by detecting the level ofenergy transfer. When two entities come close enough to each other,excitation of the donor by an energy source triggers an energy transfertowards the acceptor, which in turn emits specific fluorescence at agiven wavelength. Because of these spectral properties, a donor-acceptorcomplex can be detected without the need for physical separation fromthe unbound partners. The combination of time resolved (TR) measurementsof FRET allow the signal to be cleared of background fluorescence. Thisis typically done by introducing a time delay between the systemexcitation and fluorescence measurement to allow the signal to becleared of all non-specific short-lived emissions.

Using TR-FRET, here we demonstrate that TIGIT and CD226 elicited a FRETwhen expressed in the same cell, indicating molecular interaction ofthese two molecules. Briefly, COS-7 cells were transfected withSNAP-tagged (ST) CD226 and HA-TIGIT using Lipofectamine 2000 (LifeTechnologies) and seeded in a white 96-well plate (Costar) at 100,000cells per well. 24 hours later, cells were labeled with 100 nM ofdonor-conjugated benzyl-guanine SNAP-Lumi-4Tb (Cisbio) and 1 μMdonor-conjugated benzyl-guanine SNAP-A647 (New England Biolabs) dilutedin DMEM 10% FCS for 1 h at 37° C., 5% CO2. After three washes in PBS,the FRET signal was recorded at 665 nm for 400 μs after a 60 μs delayfollowing laser excitation at 343 nm using a Safire2 plate reader(Tecan). When the anti-TIGIT antibody was tested, the FRET signal wasalso recorded after a 15 min incubation. The FRET ratio was thencalculated as the FRET intensity divided by the donor emission at 620nm. The FRET intensity being: (signal at 665 nm from cells labeled withSNAP-donor and acceptor)—(signal at 665 nm from the same batch oftransfected cells labeled with SNAP-donor only).

As shown in FIG. 17A-17F, TIGIT was able to directly disrupt and causedissociation of CD226 homodimers. As shown in FIG. 17A-17B, thedissociation of Flag-ST-CD226 homodimers was observed with increasingconcentrations of HA-TIGIT as illustrated by the decreasing FRET ratiobetween Flag-ST-CD226 measured on COS-7 cells expressing a constantamount of Flag-ST-CD226 and increasing concentrations of HA-TIGIT.However, as shown in FIG. 17C, when anti-TIGIT antibody was added to thecell culture, this blocked the ability of TIGIT and CD226 to associate.This is illustrated by the lack of a decrease in the FRET intensity ofFlag-ST-CD226 homodimers. This demonstrates that CD226 and TIGIT areassociated as complexes but that anti-TIGIT antibodies can disrupt theseinteractions (FIG. 17A-17C).

Using TR-FRET, the ability of TIGIT to associate with CD226 was alsodemonstrated and shown in FIG. 17D-17F. Briefly, after SNAP-tag labelingusing 1 μM of donor-conjugated benzyl-guanine SNAP-A647 (see above),cells were washed three times in PBS and incubated with 2 nM of anti-HAdonor-conjugated Lumi-4Tb (Cisbio) diluted in PBS+0.2% BSA for 2 hoursat room temperature. The FRET signal was then recorded. In that case,the FRET intensity is: (signal at 665 nm from cells labeled withSNAP-acceptor and anti-HA donor)−(signal at 665 nm from mock transfectedcells labeled with SNAP-acceptor and anti-HA donor).

As shown in FIG. 17D-17E, association of Flag-ST-CD226 with HA-TIGIT wasobserved as illustrated by the increasing FRET intensity betweenFlag-ST-CD226 and HA-TIGIT measured on COS-7 cells expressing a constantamount of Flag-ST-CD226 and increasing concentrations of HA-TIGIT. Whenanti-TIGIT antibody was added, the FRET intensity decreased betweenFlag-ST-CD226 with HA-TIGIT, as shown in FIG. 17F, suggesting that theinteraction of TIGIT with CD226 can be blocked by an anti-TIGIT blockingantibody.

To confirm the cell surface expression of Flag-ST-CD226 and HA-TIGIT inthe FRET experiments, anti-Flag and anti-HA ELISA on intact COS-7 cellsexpressing the indicated tagged-constructs was performed. Briefly, COSTcells were fixed with 4% paraformaldehyde, washed twice, and blocked inphosphate-buffered saline+1% fetal calf serum (FCS). Cells were thenincubated with an anti-HA monoclonal antibody (clone 3F10, Roche appliedscience) or anti-Flag-M2 monoclonal antibody (Sigma), both conjugatedwith horseradish peroxidase. After washes, cells were incubated with aSuperSignal ELISA substrate (Pierce) and chemoluminescence was detectedon a Safire2 plate reader (Tecan). Specific signal was calculated bysubtracting the signal recorded on mock transfected cells. Asillustrated in FIG. 18, cell surface expression of both CD226 and TIGITwere confirmed in the ELISA assay.

To confirm that the TIGIT:CD226 interaction is not driven by PVRbinding, Flag-ST-CD226 and HA-TIGIT (WT) or HA-TIGIT Q56R were generatedas described in Stengel et al., (2012) PNAS 109(14):5399-5904 and FRETratios were determined as described. As shown in FIG. 24, WT TIGIT andQ56R TIGIT bind CD226 with the same efficacy.

This data not only demonstrates the CD226 and TIGIT are associated ascomplexes, but that an anti-TIGIT antibody can disrupt theseinteractions and that the TIGIT:CD226 interaction is not driven by PVRbinding. The data supports a role for TIGIT in limiting CD226-mediatedactivation of T cells and that interference with CD226 activity may bean important mechanism of action by which TIGIT inhibits T cellresponses and activity.

Example 10 CD226 Blockade Reverses the Effectors of TIGIT/PD-L1 BlockadeIn Vivo

To test the physiological relevance of the CD226 and TIGIT interaction,mice were chronically infected with Clone 13 LCMV and then treated withanti-TIGIT+ anti-PD-L1 in the absence or presence of anti-CD226 blockingantibodies. Briefly, C57BL6/J mice were briefly depleted of CD4⁺ T cellsand infected with Clone 13 strain LCMV. For chronic infections, micewere intravenously infected with 2×10⁶ PFU Clone 13 strain LCMV andtreated with 500 ug and 250 ug of depleting anti-CD4 antibodies (cloneGK1.5) 3 days before and 4 days after infection, respectively. Whereindicated, mice infected with Clone 13 strain LCMV receivedintraperitoneal injections of 200 ug of isotype control antibodies, 500ug of anti-CD226 antibodies, 200 ug of anti-PD-L1 antibodies+500 ug ofanti-TIGIT antibodies, or 500 ug of anti-CD226 antibodies+200 ug ofanti-PD-L1 antibodies+500 ug of anti-TIGIT antibodies 3 times per weekfrom days 28 to 42 post-infection. Treatment was started at 28 dayspost-infection because the T cell response is largely exhausted at thistime-point in this model of chronic viral infection (Wherry et al,Molecular Signature of CD8+ T cell Exhaustion During Chronic ViralInfection, Immunity. 2007 October; 27(4):670-84). Splenocytes and liverviral titers were analyzed 42 days after infection.

In these mice, anti-CD226 treatment alone had limited effects on CD8⁺ Tcell frequency, activation, or cytokine competency (FIG. 19A-19C).However, anti-CD226 treatment potently reversed the increases in CD8⁺ Tcell activation and IFNg production seen in mice treated withanti-PD-L1+ anti-TIGIT (59% and 58% decreases, respectively, P<0.001.FIG. 19B-19D).

Consistent with these results, LCMV viral loads were significantlyhigher in mice treated with anti-CD226+ anti-PD-L1+ anti-TIGIT than inmice treated with anti-PD-L1+ anti-TIGIT alone (272% increase, P<0.001,FIG. 19D).

This data suggests that a primary mechanism by which TIGIT limitschronic T cell responses is interference with CD226-mediatedco-stimulation. The data identifies a previously unknown role for TIGITin interacting with and disrupting CD226, resulting in the reduction orloss of a key co-stimulatory signal in CD8⁺ T cells. The datademonstrates that interference with CD226-mediated T cell costimulationmay be a major mechanism by which TIGIT limits chronic T cell responsessuch as during cancer or chronic viral infection. The data also definesan essential parameter for anti-TIGIT antibodies intended to restore theeffector function of chronically stimulated or exhausted CD8⁺ or CD4⁺ Tcells by interfering with TIGIT's ability to interact with CD226 and/orTIGIT's ability to disrupt CD226 dimerization.

Materials and Methods

Mice.

C57BL/6J and BALB/c mice were purchased from the Jackson Laboratory andCharles River Laboratories. CD4^(cre) mice and TIGIT^(loxP/loxP) micewere generated on a C57BL/6J background with standard techniques andcrossed. TIGIT expression was ablated with 96% efficiency from T cellsin TIGIT^(loxP/loxP) CD4^(cre) mice.

Flow Cytometry.

Single cell suspensions of spleen, lymph node, and tumor were preparedwith gentle mechanical disruption. Surface staining was performed withcommercial antibodies against CD4, CD8, CD44, CD62L, PD-1 (eBiosciences)and CD226 (Biolegend). TIGIT antibodies were generated at Genentech aspreviously described (Yu, X. et al. The surface protein TIGIT suppressesT cell activation by promoting the generation of mature immunoregulatorydendritic cells. Nature immunology 10, 48-57 (2009)) and conjugated toAlexa Fluor 647 according to the manufacturer's directions (MolecularProbes).

For intracellular cytokine staining (ICS), cells were stimulated for 4hours with 20 ng/mL Phorbol 12-myristate 13-acetate (PMA, Sigma) and 1μM Ionomycin (Sigma) in the presence of 3 μg/mL Brefeldin A(eBiosciences). After stimulation, cells were stained for surfacemarkers as described and fixed and permeabilized with eBioscience'sFoxP3 fixation buffer set according to the manufacturer's directions.Fixed cells were stained with antibodies against IFNγ and TNFα(eBiosciences).

Blocking Antibodies.

A blocking anti-TIGIT IgG2a monoclonal antibody (clone 10A7, reactiveagainst both mouse and human TIGIT) was generated as previouslydescribed and cloned onto a murine IgG2a backbone. A blocking anti-PD-L1IgG2a monoclonal antibody (clone 25A1) was generated by immunizingPdl1^(−/−) mice with a PD-1-Fc fusion protein and cloned onto a murineIgG2a backbone. Clone 25A1 was modified with previously describedmutations abolishing binding to Fcγ receptors. A blocking anti-CD226IgG2a monoclonal antibody (clone 37F6) was generated by immunization ofhamsters with recombinant murine CD226 and cloned onto a murine IgG2abackbone. These antibodies were also used in tests described in otherExamples described herein.

Viral Infections.

For acute infections, mice were intravenously infected with 2×10⁶plaque-forming units (PFU) Armstrong strain LCMV. For chronicinfections, mice were intravenously infected with 2×10⁶ PFU Clone 13strain LCMV and treated with 500 ug and 250 ug of depleting anti-CD4antibodies (clone GK1.5) 3 days before and 4 days after infection,respectively. Where indicated, mice infected with Clone 13 strain LCMVreceived intraperitoneal injections of 200 ug of isotype controlantibodies, 200 ug of anti-PD-L1 antibodies, and/or 500 ug of anti-TIGITantibodies 3 times per week from days 28 to 42 post-infection.

Viral Titer Assay.

Monolayers of MC57 cells were cultured with an overlay of 1%methylcellulose and infected with serially diluted liver homogenatesfrom LCMV-infected mice. 72 hours after infection, the cells were fixedwith 4% paraformaldehyde and permeabilized with 0.5% Triton-X. Viralplaques were stained with anti-LCMV NP (clone VL-4) and HRP-conjugatedanti-rat IgG and visualized with 0-phenylenediamine (OPD, Sigma).

Bioinformatics.

Breast cancer gene expression data microarray data was obtained from theCancer Gene Atlas Network (Network, T.C.G.A. Comprehensive genomiccharacterization of squamous cell lung cancers. Nature 489, 519-525(2012)). Processing and normalization of microarray data were performedusing the R programming language (http://r-project.org) andBioconductor's limma package (http://bioconductor.org). Microarrayintensity values from each channel were preprocessed using thenormal+exponential background correction method, as previouslydescribed²². Corrected intensity values were then normalized usingquantiles normalization, as previously described²³. Normalized log-ratiodata was calculated by subtracting the reference channel from the testchannel for each array. Data were further filtered using a non-specificfilter, as previously described²⁴, removing probes that do not map toknown genes, and reducing the dataset to one probe per gene. Fordifferential expression analysis, moderated t-statistics were calculatedwith the limma package, as previously described (Smyth, G. K. Linearmodels and empirical bayes methods for assessing differential expressionin microarray experiments. Statistical applications in genetics andmolecular biology 3, Article3 (2004)). To evaluate correlation,Pearson's correlation coefficients were used.

CT26 Colon Carcinoma.

BALB/c were subcutaneously inoculated with 1×10⁵ CT26 colon carcinomacells suspended in matrigel (BD Biosciences) into the right unilateralthoracic flank. After two weeks, mice bearing tumors of approximately200 mm³ were randomly recruited into treatment groups receiving 35 mg/kgof isotype control antibodies, anti-PD-L1 antibodies, and/or anti-TIGITantibodies by intraperitoneal injection 3 times per week for 3 weeks.Tumors were measured 2 times per week by caliper. Animals whose tumorsbecame ulcerated/necrotic or grew larger than 2000 mm³ were euthanized.

EMT6 Breast Carcinoma.

BALB/c mice were subcutaneously inoculated in the fourth mammary fat padwith 1×10⁵ syngeneic EMT6 breast carcinoma cells in matrigel (BDBiosciences). After two weeks, mice bearing tumors of 150-200 mm³ wererandomly recruited into treatment groups receiving 35 mg/kg of isotypecontrol antibodies, anti-PD-L1 antibodies, and/or anti-TIGIT antibodiesby intraperitoneal injection 3 times per week for 3 weeks. Tumors weremeasured 2 times per week by caliper, and tumor volumes were calculatedusing the modified ellipsoid formula, ½×(length× width²). Animals whosetumors shrank to 32 mm³ or smaller were considered to be in completeresponse (CR). Animals whose tumors grew to larger than 2000 mm³ wereconsidered to have progressed and were euthanized. Animals whose tumorsbecame ulcerated prior to progression or complete response wereeuthanized and removed from the study.

CT26 Re-Challenge.

Where indicated, BALB/c mice previously inoculated with CT26 coloncarcinoma cells as described above were re-inoculated with CT26 cellsinto the left (not previously inoculated) unilateral thoracic flank.These mice were also inoculated with 1×10⁵ EMT6 breast carcinoma cellsin matrigel into the fourth mammary fat pad. Tumors were measured 2times per week. Animals whose tumors became ulcerated/necrotic or whosetotal tumor burden exceeded 2000 mm³ were euthanized.

Statistics.

Statistical tests were conducted using unpaired (paired where specified)2-tailed Student's t-tests. Error bars depict the standard error of themean.

Animal Study Oversight.

All animal studies were approved by Genentech's Institutional AnimalCare and Use Committee.

Example 11 TIGIT Expression is Elevated in Human Cancer and Correlatedwith Expression of CD8 and PD-1 and CD8+ T Cell Infiltration

Materials and Methods

Bioinformatics. Processing and analysis of RNA-sequencing data wasperformed using the R programming language (http://www.r-project.org)along with several packages from the Bioconductor project(http://www.bioconductor.org). RNA-sequencing data for cancer andmatched normal samples were obtained from the TCGA for five differentindications: breast cancer (Network, C.G.A. Comprehensive molecularportraits of human breast tumours. Nature 490, 61-70 (2012)), colonadenocarcinoma (Network, T.C.G.A. Comprehensive molecularcharacterization of human colon and rectal cancer. Nature 487, 330-337(2012)), renal clear cell carcinoma (Network, C.G.A. Comprehensivemolecular characterization of clear cell renal cell carcinoma. Nature499, 43-49 (2013)), lung squamous cell carcinoma (Network, T.C.G.A.Comprehensive genomic characterization of squamous cell lung cancers.Nature 489, 519-525 (2012)), and endometrial carcinoma (Network,T.C.G.A. Integrated genomic characterization of endometrial carcinoma.Nature 497, 67-73 (2012)).

Raw RNA-seq reads were processed using the HTSeqGenie Bioconductorpackage. Briefly, reads were aligned to the human genome (NCBI build 37)using the GSNAP algorithm (Wu, T. D. & Nacu, S. Fast and SNP-tolerantdetection of complex variants and splicing in short reads.Bioinformatics (Oxford, England) 26, 873-881 (2010)). Uniquely alignedread pairs that fell within exons were counted to give an estimate ofgene expression level for individual genes. We used the library sizeestimation from the edgeR package (Robinson, M. D., McCarthy, D. J. &Smyth, G. K. edgeR: a Bioconductor package for differential expressionanalysis of digital gene expression data. Bioinformatics (Oxford,England) 26, 139-140 (2010)) to normalize across different samples fortheir respective sequencing depths.

To derive a T cell specific gene signature, we manually curated the Tcell genes identified by the IRIS project, removing genes associatedwith cell cycle processes, genes highly expressed in other tissues, andknown co-activating and co-inhibitory receptors. This yields a 15-genesignature that is specific to T cells. To calculate the T cell geneexpression signature score in the lung squamous cell carcinoma data, wefirst performed a variance stabilizing transform on the raw count datausing the voom function from the limma Bioconductor package. We thencalculated the first eigenvector of the centered and scaledvariance-stabilized data from the 15-gene T cell signature. Thisapproach yields a robust per-sample estimate of relative T cellabundance. A linear model including the T cell signature score was thenfit for each gene, again using the limma package. We then ranked thegenes by their correlation with the T cell signature in our linearmodel, choosing only genes positively correlated with the T cellsignature. For visualizing T cell-associated genes as a heatmap, wecentered and scaled the variance-stabilized data to unit variance,allowing for comparison of genes with different average expressionlevels.

To determine the correlation between expression of TIGIT and othergenes, we normalized RNA-sequencing count data to account fordifferences in library size, using the method from the edgeRBioconductor package (Robinson, M. D., McCarthy, D. J. & Smyth, G. K.edgeR: a Bioconductor package for differential expression analysis ofdigital gene expression data. Bioinformatics (Oxford, England) 26,139-140 (2010)). We then calculated Spearman's rank correlationcoefficient on the normalized counts. We consider rho >0.75 to beindicative of strong correlation, rho ≦0.75 but >0.5 to be indicative ofmoderate correlation, and rho ≦0.5 but >0.25 to be indicative of weakcorrelation.

For calculation of TIGIT/CD3ε ratios across each indication, we firstcalculated the variance-stabilized data for each RNA-sequencing dataset. We then calculated the log 2 ratio of the variance-stabilized datafor TIGIT and CD3ε. To calculate the difference between tumor and normalsamples, we performed standard linear model analysis using standard Rfunctions. We accepted a p-value of <0.01 as evidence of a significantdifference between tumor and normal.

To identify genes associated with tumor-infiltrating T cells, we used agene signature-based approach to interrogate gene expression data fromthe Cancer Genome Atlas (TCGA) lung squamous cell carcinoma (LUSC)collection (Network, T.C.G.A. Comprehensive genomic characterization ofsquamous cell lung cancers. Nature 489, 519-525 (2012)). Using immunecell-specific gene sets defined by the Immune Response In Silico project(Abbas, A. R. et al. Immune response in silico (IRIS): immune-specificgenes identified from a compendium of microarray expression data. Genesand immunity 6, 319-331 (2005)), and the methods described above, wedeveloped a highly specific 15 gene signature. Examining the genes mosthighly associated with the T cell signature, we identified severalco-inhibitory receptors previously associated with T cell dysfunction intumors, particularly PD-1 (FIG. 21). In LUSC, expression of TIGIT andCD3ε were highly correlated, with a Spearman's rank correlationcoefficient (ρ of 0.82 (FIG. 20A-20B). Indeed, TIGIT and CD3ε expressionwere also highly correlated in many additional TCGA tumor geneexpression datasets, including colon adenocarcinoma (COAD), uterinecorpus endrometroid carcinoma (UCEC), breast carcinoma (BRCA), andkidney renal clear cell carcinoma (KIRC), with ρ ranging from 0.83 to0.94 (FIG. 20C-20J). Furthermore, expression of TIGIT was elevatedrelative to expression of CD3ε in many tumor samples, with increasedTIGIT/CD3ε ratios in LUSC, COAC, UCEC, and BRCA tumor samples comparedto matched normal tissue (116%-419% increase, FIG. 20A-20H). The ratioof TIGIT to CD3ε expression in KIRC samples was unchanged, thoughexpression of both TIGIT and CD3ε was much higher in KIRC samples thanin normal tissue samples (FIG. 20I-20J). These data indicated that TIGITexpression was up-regulated by tumor-infiltrating lymphocytes (TILs) ina broad range of solid tumors.

TIGIT has been previously described as an inhibitor of CD4+ T cellpriming, with no known function in CD8+ T cells. However, TIGITexpression in LUSC samples was highly correlated with CD8A and onlyweakly correlated with CD4 (ρ=0.77 and 0.48 respectively, FIG. 20K-20L).Expression of TIGIT was also correlated with expression of itscomplementary co-stimulatory receptor, CD226, as well as with expressionof PD-1, a key mediator of T cell suppression in tumors and during otherchronic immune responses (ρ=0.64 and 0.82 respectively, FIG. 20M-20N).Although some non-lymphocyte cell sources of these genes exist intumors, these data strongly suggested that tumor-infiltrating T cells,particularly “exhausted” CD8⁺ T cells, expressed high levels of TIGIT.

Example 12 TIGIT and PD-1 are Coordinately Expressed by Human and MurineTumor-Infiltrating Lymphocytes

Materials and Methods

Human Tumor and PBMC Samples.

Matched whole blood and fresh surgically resected tumor tissues wereobtained from Conversant Biosciences or Foundation Bio. All specimenswere obtained with written informed consent and collected using aprotocol approved by the Hartford Hospital Institutional Review Board(IRB) (NSCLC patient 1, depicted in FIG. 22A-22P) or the Western IRB(NSCLC patient 2 and CRC patient 1, depicted in FIG. 23A-23H and FIG.37A-37E). Normal adult whole blood was obtained from a healthyvolunteer. PBMCs were purified from whole blood by Ficoll gradientcentrifugation. Tumor tissues were cut into small pieces, and incubatedwith collagenase and DNAse (Roche), and disassociated using a gentleMACSDisassociator (Miltenyi).

Flow Cytometry.

Single cell suspensions of mouse spleen, lymph node, and tumor wereprepared with gentle mechanical disruption. Surface staining wasperformed with commercial antibodies against CD4, CD8, CD44, CD62L, PD-1(eBiosciences) and CD226 (Biolegend). TIGIT antibodies were generated atGenentech and conjugated to Alexa Fluor 647 according to themanufacturer's directions (Molecular Probes).

For intracellular cytokine staining (ICS), cells were stimulated for 4hours with 20 ng/mL Phorbol 12-myristate 13-acetate (PMA, Sigma) and 1μM lonomycin (Sigma) in the presence of 3 μg/mL Brefeldin A(eBiosciences). After stimulation, cells were stained for surfacemarkers as described and fixed and permeabilized with eBioscience FoxP3fixation buffer set according to the manufacturer's directions. Fixedcells were stained with antibodies against IFNγ and TNFα (eBiosciences).

Human tumor and PBMC samples were prepared as described above. Surfacestaining was performed with a viability dye (Molecular Probes),commercial antibodies against CD45 (eBiosciences), CD3, CD4, CD8, PD-1(BD Biosciences), and with anti-TIGIT antibodies prepared as describedabove.

All samples were acquired on LSR-II or LSR-Fortessa instruments (BDBiosciences) and analyzed using FlowJo software (Treestar).

To confirm up-regulation of TIGIT by tumor-infiltrating T cells, weassessed TIGIT protein expression on human non-small-cell lung carcinomatumor-infiltrating T cells, matched peripheral T cells, and normal donorperipheral T cells. Cell surface TIGIT was expressed by subsets ofNSCLC-infiltrating CD8⁺ and CD4⁺ T cells (51% and 39% respectively, FIG.22A-22F. FIG. 36A-36B further demonstrate that cell surface TIGIT wasexpressed by a large percentage of NSCLC-infiltrating CD8⁺ and CD4⁺ Tcells (58% and 28% TIGIT respectively, FIG. 36A-36B). Interestingly,peripheral CD8⁺ and CD4⁺ T cells from the NSCLC tumor donor alsoexpressed higher levels of TIGIT than did cells from healthy donors(FIG. 22A-22F and FIG. 36A-36B). Similar results were obtained with asecond set of matched NSCLC and PBMC samples and in a set of matchedcolorectal carcinoma (CRC) and PBMC samples (FIG. 23A-23H and FIG.37A-37E). Nearly all tumor-infiltrating T cells expressing high levelsof TIGIT co-expressed PD-1, consistent with the correlation betweenTIGIT and PD-1 expression described in FIG. 1A-1J (FIG. 22G-22H).

To extend our human findings into pre-clinical cancer models, wecharacterized TIGIT expression by T cells infiltrating subcutaneous CT26and MC38 colorectal tumors in wildtype BALB/c mice and C57BL6/J mice,respectively. Two weeks post-inoculation, when CT26 and MC38 tumors hadbecome established and grown to 150-200 mm³ in size, TIGIT was expressedby approximately 50% of tumor-infiltrating CD8⁺ T cells and 25% oftumor-infiltrating CD4⁺ T cells, at levels similar to those of primaryCD8⁺ T cells stimulated in vitro (FIG. 22I-22L and FIG. 26A-26L). Inboth CD8⁺ and CD4⁺ murine TILs, CD226 was constitutively expressed, andTIGIT and PD-1 expression were again tightly correlated (FIG. 22M-22P).

These results confirmed that TIGIT was highly expressed bytumor-infiltrating T cells, and that expression of TIGIT occurred inparallel with expression other co-inhibitory receptors, most notablyPD-1.

Example 13 TIGIT Suppression of CD8+ T Cells Responses is Dependent onCD226

Unlike PD-1 or CTLA-4, there is no direct biochemical evidence of a Tcell inhibitory signaling cascade initiated by TIGIT in cis. However,co-inhibitory receptors can also function by limiting the activity of acomplementary co-stimulatory receptor, such as with the suppression ofCD28 signaling by CTLA-4. Having established TIGIT as a negativeregulator of tumor-infiltrating and anti-viral CD8⁺ T cells, we askedwhether TIGIT induced T cell exhaustion indirectly via suppression ofits complementary co-stimulatory receptor, CD226, which is highlyexpressed by peripheral and tumor-infiltrating CD8⁺ T cells (FIG.27A-27C).

Wildtype BALB/c mice bearing 150-200 mm³ CT26 tumors were treated with acombination of anti-PD-L1 and anti-TIGIT antibodies in the presence orabsence of blocking anti-CD226 antibody, or with anti-CD226 alone.Treatment with anti-CD226 alone slightly accelerated tumor growth,relative to control mice, resulting in a decreased median survival of 2days (anti-CD226 alone vs. control, P=0.0118, FIG. 28A-28G). Strikingly,the addition of anti-CD226 blocking antibodies to mice treated withanti-TIGIT and anti-PD-L1 co-blockade greatly enhanced tumor growth andfully reversed the efficacy of TIGIT/PD-L1 co-blockade on tumorregression and survival (FIG. 28A-28G). A similar effect was observed onLCMV titers in chronically infected mice treated with anti-TIGIT,anti-PD-L1, and/or anti-CD226 (FIG. 19D). These data indicated thatCD226 contributed to anti-tumor and other chronic T cell responses, andthat TIGIT suppressed these responses at least in part by suppression ofCD226.

To more fully understand how TIGIT and CD226 activity affectedanti-tumor T cell responses, we tested how CD226 alone and in concertwith TIGIT influenced T cell activation, tumor infiltration, andeffector function. We analyzed tumors and tumor-draining lymph nodesfrom CT26 tumor-bearing mice treated as above for seven days. As before,co-blockade of PD-L1 and TIGIT enhanced IFNγ production of bothtumor-infiltrating and tumor-draining lymph node-resident CD8⁺ T cells(130% and 99% increase, respectively, P<0.001, FIG. 28H-28I). Blockadeof CD226 alone had no effect on IFNγ production by tumor-infiltratingand tumor-draining lymph node-resident CD8⁺ T cells, suggesting that theeffects of CD226 co-stimulation were already limited in exhausted Tcells (FIG. 28H-28J). However, CD226 blockade did impair both thefrequency and effector function of tumor-infiltrating CD8⁺ T cells inmice treated with combination anti-TIGIT and anti-PD-L1 (57% decrease,P=0.0015, FIG. 28I). Treatment with anti-CD226 had no such effect onCD8⁺ T cells residing in the tumor-draining lymph nodes, whereasanti-PD-L1 alone enhanced CD8⁺ T cell effector function, suggesting thatPD-L1 blockade was sufficient to enhance CD8⁺ T cell effector functioneven in the absence of CD226. CD226 blockade also resulted in a reducedfrequency of tumor-infiltrating CD8⁺ T cells (53% reduction, P=0.0044,FIG. 28J-28K). Taken together, these data suggested that CD226 functionsto support both the accumulation and effector function oftumor-infiltrating CD8⁺ T cells, and that TIGIT counteracts the latter.

Example 14 TIGIT Impairs CD226 Function by Directly Disrupting CD226Homodimerization

To test if TIGIT may antagonize CD226 activity in cis, TIGIT's effect onCD226 co-stimulation in vitro was tested. TIGIT-deficient CD8⁺ T cellsstimulated with sub-optimal levels of anti-CD3 responded more robustlyto PVR co-stimulation than did wildtype littermate CD8⁺ T cells, andthis enhanced response was dependent on CD226 (46% increase inproliferation, P=0.0061, FIG. 29A). Consistent with these data, wildtypeCD8⁺ T cells, stimulated with sub-optimal anti-CD3 and PVR, proliferatedmore robustly in the presence of anti-TIGIT antibodies than they did inthe presence of isotype-matched control antibodies, and this effect wasalso dependent on CD226 (105% increase in proliferation, P=0.0010, FIG.29B).

To test the relevance of TIGIT to primary human CD8⁺ T cells, wepurified CD8⁺ T cells from healthy donor blood and stimulated them withsub-optimal levels of plate-bound anti-CD3 and recombinant human PVR-Fcfusion protein. In the presence of isotype-matched control antibodies,PVR co-stimulation moderately enhanced T cell stimulation andproliferation. Furthermore, addition blocking anti-TIGIT antibodiessignificantly enhanced the effects of PVR co-stimulation, consistentwith TIGIT's effects on primary murine CD8⁺ T cells (69% increase inproliferation, P=0.0071, FIG. 29C). These data demonstrated acell-intrinsic role for TIGIT inhibition of CD226 function on primarymurine and human CD8⁺ T cells.

TR-FRET (Time-resolved Fluorescence Resonance Energy Transfer) was usedto determine the molecular mechanism by which TIGIT impaired CD226activity. First, we expressed and labeled human ST-CD226 withnon-permeant donor and acceptor fluorophores. These cells yielded astrong FRET signal, confirming the ability of CD226 to homodimerize(FIG. 29D). To monitor CD226 and TIGIT interactions on the cell surface,we expressed ST-CD226 in absence or in presence of human HA-TIGIT thatwe labeled with the SNAP-tag substrate and an anti-HA antibody,respectively. Strikingly, co-expression of increasing amounts of TIGIT(monitored by ELISA) attenuated the CD226/CD226 FRET signal, indicatingthat TIGIT could disrupt CD226 homodimerization (FIG. 29E). Indeed,acceptor CD226 and donor TIGIT also resulted in a significant FRETsignal, indicating a direct interaction between these two proteins (FIG.29F). This interaction was further confirmed by co-immunoprecipitation(FIG. 29G). These data demonstrated that TIGIT and CD226 directlyinteract at the cell surface, and that this interaction can impair CD226homodimerization.

To test the effects of TIGIT antibody blockade on TIGIT-CD226interaction, we again co-expressed human ST-CD226 and HA-TIGIT, thistime in the presence or absence of blocking antibodies against humanTIGIT. The addition of anti-TIGIT to the cell cultures significantlyreduced the ability of TIGIT and CD226 to associate (FIG. 29H). Thesedata suggested that anti-TIGIT treatment can limit TIGIT's interactionwith CD226, and are consistent with the notion that suppression of CD226activity is a key mechanism of action by which TIGIT enforces CD8⁺ Tcell exhaustion. This is also consistent with the ability of anti-TIGITantibodies to enhance CD226 co-stimulation.

Next, we confirmed the capacity of endogenous TIGIT and CD226 tointeract (FIG. 30). Primary human T cells were stimulated in vitro withanti-CD3 and anti-CD28 antibodies, sorted on the basis of TIGITexpression, rested, re-stimulated, and labeled with antibodies againstendogenous TIGIT and CD226 that were conjugated to fluorophorescompatible with TR-FRET. TIGIT-expressing T cells labeled withdonor-conjugated anti-TIGIT and acceptor-conjugated anti-CD226antibodies yielded a strong FRET signal (FIG. 30). In contrast, only anegligible FRET signal was detected on T cells that did not expressTIGIT or that were labeled with donor-conjugated anti-TIGIT andacceptor-conjugated anti-HVEM antibodies (FIG. 30), confirming thespecificity of the detected interaction between endogenous TIGIT andCD226.

These results demonstrate that endogenous TIGIT and CD226 can directlyinteract at the cell surface, and that this interaction impairs CD226homodimerization. Given the role of CD226 as a co-stimulator of T cellresponses in vivo, and without wishing to be bound by theory, it isbelieved that suppression of CD226 may be a key mechanism of action bywhich TIGIT enforces CD8⁺ T cell exhaustion during chronic viralinfection and cancer.

Materials and Methods

Time-Resolved Fluorescence Resonance Energy Transfer with TransfectedCell Lines.

CHO cells were transfected with N-terminus SNAP-tagged (ST) CD226 andN-terminus HA-TIGIT using Lipofectamine 2000 (Life Technologies) andseeded in a white 96-well plate (Costar) at 100,000 cells per well. 24hours later, cells were labeled to measure TR-FRET either betweenSNAP-donor/SNAP-acceptor or between SNAP-acceptor/anti-HA donor. 1)SNAP-donor /SNAP-acceptor labeling: Cells were incubated with 100 nM ofdonor-conjugated benzyl-guanine SNAP-Lumi-4Tb (Cisbio) and 1 μMacceptor-conjugated benzyl-guanine SNAP-A647 (New England Biolabs)diluted in DMEM 10% FCS for 1 h at 37° C., 5% CO₂. Cells were thenwashed three times in PBS before reading of the FRET signal. 2)SNAP-acceptor/anti-HA donor: Cells were incubated with 1 μMacceptor-conjugated benzyl-guanine SNAP-A647 diluted in DMEM 10% FCS for1 h at 37° C., 5% CO₂. After three washes in PBS, cells were incubatedfor 2 hours with 2 nM anti-HA Lumi-4Tb (Cisbio) in PBS+0.2% BSA at roomtemperature. The FRET signal was then recorded at 665 nm for 400 μsafter a 60 μs delay following laser excitation at 343 nm using a Safire2plate reader (Tecan). When anti-TIGIT was tested at 10 μg/ml final, theFRET signal was also recorded after a 15 min incubation. For theFlag-ST-CD226/Flag-ST-CD226 interaction, the FRET ratio was calculatedas the FRET intensity divided by the donor emission at 620 nm, which isproportional to the CD226 expression. The FRET intensity being: (signalat 665 nm from cells labeled with SNAP-donor and acceptor)−(signal at665 nm from the same batch of transfected cells labeled with SNAP-donoronly). For the Flag-ST-CD226/HA-TIGIT interaction, the FRET ratiorepresents the FRET intensity divided by the Flag-ST-CD226 expression asmeasured by an anti-Flag ELISA. In that case, the FRET intensity=(signalat 665 nm from cells labeled with SNAP-acceptor and anti-HAdonor)−(signal at 665 nm from mock transfected cells labeled withSNAP-acceptor and anti-HA donor).

Time-Resolved Fluorescence Resonance Energy Transfer with Human T Cells.

Human anti-TIGIT (Genentech clone 1F4), anti-CD226 (Santa CruzBiotechnology), and anti-HVEM (eBioscience) antibodies were conjugatedfluorophores compatible with TR-FRET (Cisbio). Primary human T cellswere MACS-enriched from blood, stimulated in vitro with plate boundanti-CD3 and anti-CD28 for 72 hours. TIGIT-expressing and non-expressingT cells (all expressing CD226) were then sorted, rested withoutstimulation for 72 hours, and re-stimulated for 48 hours. Eachpopulation was then washed once with Tris-KREBS buffer (20 mM Tris pH7.4, 118 mM NaCl, 5.6 mM glucose, 1.2 mM KH2PO4, 1.2 mM MgSO4, 4.7 mMKCl, 1.8 mM CaCl2) and cultured under the following conditions, intriplicate: 1) Anti-TIGIT Ab-Lumi4-Tb (5 μg/ml), 2) Anti-TIGITAb-Lumi4-Tb (5 μg/ml)+anti-HVEM-d2 (10 μg/ml), 3) Anti-TIGIT Ab-Lumi4-Tb(5 μg/ml)+anti-CD226 (10 μg/ml), 4) Anti-TIGIT Ab-Lumi4-Tb (5μg/ml)+anti-CD226 (10 μg/ml)+cold anti-TIGIT Ab (clone 1F4) (50 μg/ml).The indicated concentrations were optimized to ensure the highest FRETsignal. Cells were incubated for 2 hours at room temperature on arotator and then washed 3 times in Tris-KREBS buffer. T cells were thenseeded at 400,000 cells/well in a white 96-well plate (Costar) andTR-FRET was recorded at 665 nm for 400 μs after a 60 μs delay followinglaser excitation at 343 nm using a PHERAstar plate reader (BMG Labtech).FRET intensity was expressed as the signal at 665 nm from cells labeledwith Ab-Lumi4-Tb+Ab-d2 minus the signal at 665 nm from the same batch ofcells labeled with Ab-Lumi4-Tb alone. The non-specific FRET signal wasgiven by the T cells incubated with Lumi4Tb+d2+an excess of cold Ab.

Co-Immunoprecipitation.

Briefly, COS 7 Cells in 15 cm plates were co-transfected with expressionplasmids containing the cDNA for either TIGIT-HA (5 ng) or CD226-Flag(10 ng) tagged proteins, or a control plasmid (pRK). 23 hrs aftertransfection the cells were washed with PBS and harvested in 4 ml of icecold PBS and centrifuged at 300×g for 5 min and cell pellets werere-suspended in 2 ml of Lysis buffer at 4° C. The cells were lysed over50 min with vortexing every 15 min and subsequently centrifuged at10,00×g for 15 min at 4° C. The resultant supernatant was pre-clearedwith 160 μl of CL6B sepahrose slurry by rotating for 30 min at 4° C.,and centrifuged for 2 min at 3000×g. The supernatant was equally splitinto two tubes and immuno-precipitated with either an anti-HA or ananti-flag using standard procedures. The immune-precipitated proteinswere subjected to SDS-PAGE and western blotted. Western blots wereprobed with either anti-Flag-HRP or anti-HA-HRP.

All patents, patent applications, documents, and articles cited hereinare herein incorporated by reference in their entireties.

1. A method for treating or delaying progression of cancer in anindividual comprising administering to the individual an effectiveamount of a PD-1 axis binding antagonist and an agent that decreases orinhibits TIGIT expression and/or activity.
 2. (canceled)
 3. A method fortreating or delaying progression of an immune related disease in anindividual comprising administering to the individual an effectiveamount of a PD-1 axis binding antagonist and an agent that decreases orinhibits TIGIT expression and/or activity.
 4. (canceled)
 5. The methodof claim 3, wherein the immune related disease is associated with a Tcell dysfunctional disorder. 6-7. (canceled)
 8. The method of claim 5,wherein the T cell dysfunctional disorder is characterized by T cellexhaustion.
 9. (canceled)
 10. The method of claim 3, wherein the immunerelated disease is selected from the group consisting of unresolvedacute infection, chronic infection, and tumor immunity.
 11. (canceled)12. A method of treating or delaying progression of cancer in anindividual comprising administering to the individual an effectiveamount of a PD-1 axis binding antagonist and an agent that modulatesCD226 expression and/or activity.
 13. (canceled)
 14. A method fortreating or delaying progression of an immune related disease in anindividual comprising administering to the individual an effectiveamount of a PD-1 axis binding antagonist and an agent that modulatesCD226 expression and/or activity.
 15. (canceled)
 16. The method of claim14, wherein the immune related disease is associated with a T celldysfunctional disorder. 17-18. (canceled)
 19. The method of claim 16,wherein the T cell dysfunctional disorder is characterized by T cellexhaustion.
 20. (canceled)
 21. The method of claim 14, wherein theimmune related disease is selected from the group consisting ofunresolved acute infection, chronic infection and tumor immunity.
 22. Amethod of increasing, enhancing, or stimulating an immune response orfunction in an individual comprising administering to the individual aneffective amount of a PD-1 axis binding antagonist and an agent thatmodulates CD226 expression and/or activity. 23-25. (canceled)
 26. Themethod of any one of claims 12, 14, and 22, wherein the agent thatmodulates CD226 expression and/or activity is selected from the groupconsisting of an agent that inhibits and/or blocks the interaction ofCD226 with TIGIT, an antagonist of TIGIT expression and/or activity, anantagonist of PVR expression and/or activity, an agent that inhibitsand/or blocks the interaction of TIGIT with PVR, an agent that inhibitsand/or blocks the interaction of TIGIT with PVRL2, an agent thatinhibits and/or blocks the interaction of TIGIT with PVRL3, an agentthat inhibits and/or blocks the intracellular signaling mediated byTIGIT binding to PVR, an agent that inhibits and/or blocks theintracellular signaling mediated by TIGIT binding to PVRL2, an agentthat inhibits and/or blocks the intracellular signaling mediated byTIGIT binding to PVRL3, and combinations thereof.
 27. (canceled)
 28. Themethod of claim 26, wherein the agent that inhibits and/or blocks theinteraction of CD226 with TIGIT is a small molecule inhibitor, aninhibitory antibody or antigen-binding fragment thereof, an aptamer, aninhibitory nucleic acid, or an inhibitory polypeptide.
 29. The method ofclaim 28, wherein the inhibitory antibody or antigen-binding fragmentthereof is an anti-TIGIT antibody or antigen-binding fragment thereof.30. (canceled)
 31. The method of claim 26, wherein the agent thatmodulates CD226 expression and/or activity is an antagonist of TIGITexpression and/or activity.
 32. The method of claim 31, wherein theantagonist of TIGIT expression and/or activity is a small moleculeinhibitor, an inhibitory antibody or antigen-binding fragment thereof,an aptamer, an inhibitory nucleic acid, or an inhibitory polypeptide.33. The method of claim 32, wherein the inhibitory antibody orantigen-binding fragment thereof is an anti-TIGIT antibody orantigen-binding fragment thereof. 34-41. (canceled)
 42. A method ofincreasing, enhancing, or stimulating an immune response or function inan individual comprising administering to the individual an effectiveamount of an agent that decreases or inhibits TIGIT expression and/oractivity and an agent that decreases or inhibits one or more additionalimmune co-inhibitory receptors.
 43. The method of claim 42, wherein theone or more additional immune co-inhibitory receptor is selected fromthe group consisting of PD-1, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, andCD96. 44-48. (canceled)
 49. The method of any one of claims 12, 14, 22,and 42, further comprising administering at least one chemotherapeuticagent.
 50. The method of claim 22 or 42, wherein the individual hascancer. 51-58. (canceled)
 59. The method of claim 12, wherein the cancerhas elevated levels of T cell infiltration. 60-70. (canceled)
 71. Themethod of claim 29, wherein the anti-TIGIT antibody or antigen-bindingfragment thereof comprises at least one HVR comprising an amino acidsequence selected from the amino acid sequences (1) KSSQSLYYSGVKENLLA(SEQ ID NO:1), ASIRFT (SEQ ID NO:2), QQGINNPLT (SEQ ID NO:3), GFTFSSFTMH(SEQ ID NO:4), FIRSGSGIVFYADAVRG (SEQ ID NO:5), and RPLGHNTFDS (SEQ IDNO:6); or (2) RSSQSLVNSYGNTFLS (SEQ ID NO:7), GISNRFS (SEQ ID NO:8),LQGTHQPPT (SEQ ID NO:9), GYSFTGHLMN (SEQ ID NO:10), LIIPYNGGTSYNQKFKG(SEQ ID NO:11), and GLRGFYAMDY (SEQ ID NO:12).
 72. The method of claim71, wherein the anti-TIGIT antibody or antigen-binding fragment thereofcomprises a light chain comprising the amino acid sequence set forth inDIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKR (SEQID NO:13) orDVVLTQTPLSLSVSFGDQVSISCRSSQSLNNSYGNTFLSWYLHKPGQSPQLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPPTFGPGTKLEVK (SEQ ID NO:14).
 73. The methodof claim 71, wherein the anti-TIGIT antibody or antigen-binding fragmentthereof comprises a heavy chain comprising the amino acid sequence setforth inEVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHVVRQSPGKGLEWVVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSS (SEQ ID NO:15) orEVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVVKQSHGKNLEWIGLIIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGLRGFYAMDYWGQGTSVTVSS (SEQ ID NO:16).
 74. Themethod of claim 71, wherein the anti-TIGIT antibody or antigen-bindingfragment thereof comprises a light chain comprising the amino acidsequence set forth in DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKR (SEQID NO:13) orDVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPPTFGPGTKLEVK (SEQ ID NO:14), and a heavychain comprising the amino acid sequence set forth inEVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSS (SEQ ID NO:15) orEVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGLIIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGLRGFYAMDYWGQGTSVTVSS (SEQ ID NO: 16). 75.The method of claim 29, wherein the anti-TIGIT antibody orantigen-binding fragment thereof is selected from the group consistingof a humanized antibody, a chimeric antibody, a bispecific antibody, aheteroconjugate antibody, and an immunotoxin.
 76. The method of claim29, wherein the anti-TIGIT antibody or antigen-binding fragment thereofcomprises at least one HVR that is at least 90% identical to an HVR setforth in any one of (1) KSSQSLYYSGVKENLLA (SEQ ID NO:1), ASIRFT (SEQ IDNO:2), QQGINNPLT (SEQ ID NO:3), GFTFSSFTMH (SEQ ID NO:4),FIRSGSGIVFYADAVRG (SEQ ID NO:5), and RPLGHNTFDS (SEQ ID NO:6); or (2)RSSQSLVNSYGNTFLS (SEQ ID NO:7), GISNRFS (SEQ ID NO:8), LQGTHQPPT (SEQ IDNO:9), GYSFTGHLMN (SEQ ID NO:10), LIIPYNGGTSYNQKFKG (SEQ ID NO:11), andGLRGFYAMDY (SEQ ID NO:12).
 77. (canceled)
 78. The method of any one ofclaims 12, 14, and 22, wherein the PD-1 axis binding antagonist isselected from the group consisting of a PD-1 binding antagonist, a PD-L1binding antagonist and a PD-L2 binding antagonist. 79-88. (canceled) 89.The method of claim 78, wherein the PD-1 axis binding antagonist is aPD-L1 binding antagonist.
 90. The method of claim 89, wherein the PD-L1binding antagonist inhibits the binding of PD-L1 to PD-1. 91-92.(canceled)
 93. The method of claim 89, wherein the PD-L1 bindingantagonist is an anti-PD-L1 antibody.
 94. The method of claim 93,wherein the anti-PD-L1 antibody is selected from the group consisting ofYW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736.
 95. The method of claim93, wherein the anti-PD-L1 antibody comprises a heavy chain comprisingHVR-H1 sequence of GFTFSDSWIH (SEQ ID NO:17), HVR-H2 sequence ofAWISPYGGSTYYADSVKG (SEQ ID NO:18), and HVR-H3 sequence of RHWPGGFDY (SEQID NO:19); and a light chain comprising HVR-L1 sequence of RASQDVSTAVA(SEQ ID NO:20), HVR-L2 sequence of SASFLYS (SEQ ID NO:21), and HVR-L3sequence of QQYLYHPAT (SEQ ID NO:22).
 96. The method of claim 95,wherein the anti-PD-L1 antibody comprises a heavy chain variable regioncomprising the amino acid sequence ofEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA (SEQ ID NO:23),EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK (SEQ ID NO:40), orEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO:41), and alight chain variable region comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKWYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO:24). 97-99. (canceled)100. The method of claim 12, wherein the cancer is selected from thegroup consisting of non-small cell lung cancer, small cell lung cancer,renal cell cancer, colorectal cancer, ovarian cancer, breast cancer,pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer,mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma,prostate cancer, glioblastoma, cervical cancer, thymic carcinoma,leukemia, lymphomas, myelomas, mycoses fungoids, merkel cell cancer, andother hematologic malignancies. 101-120. (canceled)
 121. A kitcomprising a PD-1 axis binding antagonist and a package insertcomprising instructions for using the PD-1 axis binding antagonist incombination with an agent that modulates CD226 expression and/oractivity to treat or delay progression of cancer in an individual.122-123. (canceled)
 124. A kit comprising a PD-1 axis binding antagonistand a package insert comprising instructions for using the PD-1 axisbinding antagonist in combination with an agent that modulates CD226expression and/or activity to enhance immune function of an individualhaving cancer. 125-136. (canceled)
 137. A kit comprising an agent thatdecreases or inhibits TIGIT expression and/or activity and a packageinsert comprising instructions for using the agent that decreases orinhibits TIGIT expression and/or activity in combination with an agentthat decreases or inhibits one or more additional immune co-inhibitoryreceptors to treat or delay progression of cancer in an individual.138-139. (canceled)
 140. A kit comprising an agent that decreases orinhibits TIGIT expression and/or activity and a package insertcomprising instructions for using the agent that decreases or inhibitsTIGIT expression and/or activity in combination with an agent thatdecreases or inhibits one or more additional immune co-inhibitoryreceptors to enhance immune function of an individual having cancer.141-153. (canceled)
 154. The kit of claim 137 or 140, wherein the agentthat decreases or inhibits TIGIT expression and/or activity is selectedfrom the group consisting of an antagonist of TIGIT expression and/oractivity, an antagonist of PVR expression and/or activity, an agent thatinhibits and/or blocks the interaction of TIGIT with PVR, an agent thatinhibits and/or blocks the interaction of TIGIT with PVRL2, an agentthat inhibits and/or blocks the interaction of TIGIT with PVRL3, anagent that inhibits and/or blocks the intracellular signaling mediatedby TIGIT binding to PVR, an agent that inhibits and/or blocks theintracellular signaling mediated by TIGIT binding to PVRL2, and an agentthat inhibits and/or blocks the intracellular signaling mediated byTIGIT binding to PVRL3.
 155. The kit of claim 154, wherein theantagonist of TIGIT expression and/or activity is an anti-TIGIT antibodyor antigen-binding fragment thereof. 156-158. (canceled)
 159. The kit ofclaim 121 or 124, wherein the agent that modulates CD226 expressionand/or activity is selected from the group consisting of an agent thatinhibits and/or blocks the interaction of CD226 with TIGIT, anantagonist of TIGIT expression and/or activity, an antagonist of PVRexpression and/or activity, an agent that inhibits and/or blocks theinteraction of TIGIT with PVR, an agent that inhibits and/or blocks theinteraction of TIGIT with PVRL2, an agent that inhibits and/or blocksthe interaction of TIGIT with PVRL3, an agent that inhibits and/orblocks the intracellular signaling mediated by TIGIT binding to PVR, anagent that inhibits and/or blocks the intracellular signaling mediatedby TIGIT binding to PVRL2, and an agent that inhibits and/or blocks theintracellular signaling mediated by TIGIT binding to PVRL3. 160.(canceled)
 161. The kit of claim 159, wherein the agent that inhibitsand/or blocks the interaction of CD226 with TIGIT is a small moleculeinhibitor, an inhibitory antibody or antigen-binding fragment thereof,an aptamer, an inhibitory nucleic acid, or an inhibitory polypeptide.162. The kit of claim 161, wherein the inhibitory antibody orantigen-binding fragment thereof is an anti-TIGIT antibody orantigen-binding fragment thereof.
 163. The kit of claim 155, wherein theanti-TIGIT antibody or antigen-binding fragment thereof comprises atleast one HVR comprising an amino acid sequence selected from the aminoacid sequences (1) KSSQSLYYSGVKENLLA (SEQ ID NO:1), ASIRFT (SEQ IDNO:2), QQGINNPLT (SEQ ID NO:3), GFTFSSFTMH (SEQ ID NO:4),FIRSGSGIVFYADAVRG (SEQ ID NO:5), and RPLGHNTFDS (SEQ ID NO:6); or (2)RSSQSLVNSYGNTFLS (SEQ ID NO:7), GISNRFS (SEQ ID NO:8), LQGTHQPPT (SEQ IDNO:9), GYSFTGHLMN (SEQ ID NO:10), LIIPYNGGTSYNQKFKG (SEQ ID NO:11), andGLRGFYAMDY (SEQ ID NO:12). 164-165. (canceled)
 166. The kit of claim163, wherein the anti-TIGIT antibody or antigen-binding fragment thereofcomprises a light chain comprising the amino acid sequence set forth inDIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKR (SEQID NO:13) orDVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPPTFGPGTKLEVK (SEQ ID NO:14), and a heavychain comprising the amino acid sequence set forth inEVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSS (SEQ ID NO:15) orEVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGLIIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGLRGFYAMDYWGQGTSVTVSS (SEQ ID NO: 16). 167.The kit of claim 162, wherein the anti-TIGIT antibody or antigen-bindingfragment thereof comprises at least one HVR comprising an amino acidsequence selected from the amino acid sequences (1) KSSQSLYYSGVKENLLA(SEQ ID NO:1), ASIRFT (SEQ ID NO:2), QQGINNPLT (SEQ ID NO:3), GFTFSSFTMH(SEQ ID NO:4), FIRSGSGIVFYADAVRG (SEQ ID NO:5), and RPLGHNTFDS (SEQ IDNO:6); or (2) RSSQSLVNSYGNTFLS (SEQ ID NO:7), GISNRFS (SEQ ID NO:8),LQGTHQPPT (SEQ ID NO:9), GYSFTGHLMN (SEQ ID NO:10), LIIPYNGGTSYNQKFKG(SEQ ID NO:11), and GLRGFYAMDY (SEQ ID NO:12).
 168. The kit of claim167, wherein the anti-TIGIT antibody or antigen-binding fragment thereofcomprises a light chain comprising the amino acid sequence set forth inDIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKR (SEQID NO:13) orDVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPPTFGPGTKLEVK (SEQ ID NO:14), and a heavychain comprising the amino acid sequence set forth inEVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSS (SEQ ID NO:15) orEVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGLIIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGLRGFYAMDYWGQGTSVTVSS (SEQ ID NO: 16)